**Plant Defense Enzymes Activated in Bean Plants by Aqueous Extract from**  *Pycnoporus sanguineus* **Fruiting Body**

José Renato Stangarlin, Clair Aparecida Viecelli, Odair José Kuhn, Kátia Regina Freitas Schwan-Estrada, Lindomar Assi, Roberto Luis Portz and Cristiane Cláudia Meinerz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50686

## **1. Introduction**

152 Plant Science

79p.

Cowling, E.D. 1961. Comparative Biochemistry of the decay of sweet gum sapwood by white rot and brown rot fungi. *U.S. Department of Agriculture, Technical Bulletin* No. 1258,

Darvill , A.G., and Albersheim, P. 1984. Phytoalexins and their elicitors – A defence against

Geiger, J.P., Rio, B., Nicole, M., and Nandris, D. 1986. Biodegradation of *Hevea Brasiliensis* wood by *Rigidoporus lignosus* and *Phellinus noxius*. *Eur.J . For. Path;* 16:147-159. Gianinazzi, S.1984. Genetic and molecular aspects of resistance induced by infections or chemicals. In : *Plant –microbe Interactions Molecular and genetic perspectives*. Vol 1. (Eds;

Igeleke, C.L. 1988. Diseases of Rubber ( *Hevea brasiliensis)* and their control. Paper presented at the 10th Annual conference of the Horticultural society of Nigeria, 6-8 Nov. 1988 Jacob, C.K 2006. Corynespora leaf disease of *Hevea brasiliensis:* A threat to natural rubber production (9-16pp) in Corynespora leaf disease of *H. brasiliensis:* strategies for

Jayasuriya, K.E. 2004. Factors affecting disease tolerance of rubber tree and research needs for developing disease tolerant genotypes for the sustainability of rubber industry.

Jayaratne, R, Wettasinghe, P.C., Siriwardene, D., and Peiris D. 2001. Systemic fungicides as a drench application to control white root disease of rubber. *Journal of the Rubber Research* 

Jones, D. H. 1984. Phenylanine ammonia – lyase: regulation of its induction, and its role in

Kirk, T.K . 1971. Effect of microorganisms on ligin. *Annual Review of Phytopathology,* 9:185-210. Malaysian Rubber Board. 2000. *Treatment of maladies and injuries and control of pests.* In: Rubber Plantation and Processing technologies. Malaysian Rubber Board, 2000. Nandris, D., Nicole, M. and Geiger, J.P. 1987. Root Rot Diseases of Rubber Trees. *Plant* 

Narasimhan, K.T and Kothandaraman, R. 2000. Detection of pathogenesis related proteins in *Hevea brasiliensis* infected by *Phytophthora meadii*. *Indian Journal of Natural Rubber*, 13:30-37. Nicholson, R.L,. and Hammerschmidt, R. 1992. Phenolic compounds and their role in

Nicole, M., Geiger, J.P., and Nandris, D. 1985. Variability among Africa populations of

Otoide, V.O. 1978. Further observations on the pre-treatment of forest trees for root disease

Rajalakshmy, V.K, and Jayarathnam, K.2000. Root diseases and non- microbial maladies. In: *Natural Rubber: Agro-management and crop processing* (Eds. P.J George and C. Kuruvilla

Simons, T.J., and Rose, A.F. 1971. Metabolic changes associated with systemic induced resistance to tobacco mosaic virus in Samsun -NN tobacco. *Phytopathology,* 61:293 -300.

microbial infection in plants. *Annual Review Plant Physiology* 35:243-275.

Kosuge, T and Nester, E.W), 321-342 PP. Macmillan, New York.

management (Ed. C.K. Jacob). Rubber Research institute of India.

*Bulletin of the Rubber institute of Sri Lanka,* 45 : 1-10.

plant development. *Phytochemistry,* 23:1349-1369.

disease resistance. *Annual Review of Pyhtopathology* 30:369-389.

*Rigidoporus lignosus and Phellinus noxius, Eur. J. for. Pathol*. 15: 293 -300.

Jacob) Rubber Research Institute of India, Kohayam, Kerala, India.

control in *Hevea* plantings. Paper presented at RRIN Seminar, 1978, 7Pp.

*Institute of Srilanka*, 84:1-17.

*Disease,* 71 (4) : 298-306.

The common bean (*Phaseolus vulgaris* L.) can be affected for more than 300 diseases caused by virus, bacteria, fungi, and nematodes. The semibiotroph *Pseudocercospora griseola* (Sacc.) Crous & Braun (sin. *Phaeoisariopsis griseola* (Sacc.) Ferraris), the causal agent of angular leaf spot, represents one of the main fungal pathogens of this crop, manifesting on the stem, leaf, and pod [1].

Traditionally, the control of the angular leaf spot has been done with the use of resistant cultivars, seeds free of pathogen and fungicides. The last one, at a short time, has it's advantages, but for a long period of time, can cause problems due the residues accumulation and environmental pollution [2]. Thus, with the objective to find new technologies, ecologically or environmentally safer, for the control of plant diseases, mainly in organic growth, alternative methods for the control of phytopathogens are been development. This kind of alternative methods are been investigated by our 'Biological and Alternative Control of Plant Diseases' research group [3].

The induction of resistance in plants involves the activation of defense latent mechanisms [4] in response to the treatment with elicitor agents, protecting against subsequent infection by pathogens. Among the non-conventional elicitors can be included the extracts of medicinal plants and essential oils [5], homeopathic drugs [6], as well as the extracts obtained from mushrooms [7-9]. Among the basidiomycetes with elicitor proprieties stands out *Pycnoporus sanguineus* (L. ex Fr.) Murr. [10], utilized since the medicine [11,12] to the alternative control of plant diseases [13,14]. These previous works had shown that the biological properties of

© 2012 Stangarlin et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Stangarlin et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*P. sanguineus* depending of its crude or aqueous extract and not of its individual compounds, like cinnabarin, or extracts obtained from organic solvents.

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 155

**Inhibition of conidia germination:** This assay was done in microscopic slide covered with a thin layer of agar-water 1% (700 L per slide) [20]. Aliquots of 40 L of aqueous extracts in concentrations 0, 1, 5, 10, 15 and 20%, sterilized in autoclave or filtrated in nitrocellulose membrane (0.45 m of pore diameter) and aliquots of 40 L of conidia suspension of *P. griseola* (1x104 conidia mL-1) obtained of a culture with 14 days old, were distributed in the surface of the slide, which were incubated in moist chamber under dark at 24 °C [21]. As control treatments were utilized fungicide (azoxystrobin: 40 mg L-1) and acibenzolar-Smethyl (ASM: 75 mg L-1). The percentage of the germination was determined after 24 hours with the addition of 40 L of lactophenol cotton-blue in each slide to paralyze the conidia

**Inhibition of the mycelial growth and sporulation of** *P. griseola***:** The extracts of basidiocarp of *P. sanguineus* were incorporated in concentrations of 0, 1, 5, 10, 15 and 20%, in tomato juice culture medium. The extracts were sterilized in autoclave and also by filtration in nitrocellulose membrane (0.45 m of diameter of pore) [22]. As control treatments were utilized fungicide (azoxystrobin: 40 mg L-1) and acibenzolar-S-methyl (ASM: 75 mg L-1). For transferring *P. griseola* to Petri dishes, 100 µL of spore suspension (1x104 conidia mL-1) were added to the medium and homogenized with Drygalski loop. The Petri dishes were sealed with plastic film and maintained at 24 °C and dark. Were evaluated the diameter and the number of colonies 14 days after the beginning of the experiment. At the end of the assay of mycelial growth inhibition, it was evaluated the sporulation of the fungus. For this, was prepared a suspension of conidia by the addition of 10 mL of distillated water per plate and

**Experiment in greenhouse:** Two plants of common bean (cultivar IAPAR 81 – Carioca) were cultivated in plastic pots containing 5 L of a mixture of sterilized soil and sand (proportion 2:1). To resistance induction assay, were used aqueous extract of *P. sanguineus* basidiocarp at concentration of 10% and 20%. As control treatments were used water, fungicide (azoxystrobin - 40 mg L-1) and acibenzolar-S-methyl (75 mg L-1). The extracts and the control

**Field experiment:** The experiment consisted in three randomized blocks, with five plots per block. Each plot consisted of three lines of 3 m of length, spaced 0.5 m between them, with 10 plants (cultivar IAPAR 81 – Carioca) per meter. The central line, discounting 0.5 m from the anterior and posterior borders, was considerate as useful area for evaluation. For the assay of resistance induction, were sprayed aqueous extracts of *P. sanguineus* basidiocarp at concentrations of 10% and 20%, and as control treatments were used water, fungicide (azoxystrobin: 40 mg L-1) and the acibenzolar-S-methyl (ASM: 75 mg L-1). The extracts (5 mL per plant) were applied twice, the first one in vegetative stage (V3) and the second in

**Pathogen inoculation:** The conidia suspension of *P. griseola* was prepared in water with Tween 20 (one drop 500 mL-1), and the concentration adjusted to 4x104 conidian mL-1. The inoculation in the greenhouse was done three days after the application of extracts and control treatments, in the 3rd treated leaf, as well as in the 4th non-treated leaf (vegetative

determined the number of conidian per mL in Neubauer chamber.

treatments were sprayed in the 3rd leaf (vegetative stage V4) (3 mL per leaf).

germination.

reproductive stage (R3).

Previous works had shown the potential of *P. sanguineus* components for controlling plant diseases. Aqueous extracts, obtained from liquid medium-culture filtrate (MCF) [15] and from mycelium (AEM) [16] of *P. sanguineus*, were capable to reduce in 82% and 49% to MCF, and in 93% and 50% to AEM, in greenhouse and field conditions, respectively, the severity of angular leaf spot in bean plants. However, the effect of *P. sanguineus* fruiting bodies in that pathosystem was not investigated. Against plant pathogenic bacteria, fruiting body extracts from *P. sanguineus* were efficient for the control of common bacterial blight in bean, caused by *Xanthomonas axonopodis* pv. *phaseoli* (Smith – Vauterin, Hoste, Kersters & Swings) which can occur either by direct antimicrobial activity and by resistance induction involving the activation of some pathogenesis-related proteins [17].

In another experiment, the *in situ* detection of reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2) and superoxide (O2.-), was searched in bean plants treated with aqueous extracts of the mycelium (AEM) and basidiocarps or fruiting bodies (AEB) of *P. sanguineus* and inoculated after three days with *Colletotrichum lindemuthianum* ((Sacc. & Magn.) Scrib.). It was possible to detect H2O2 at 48 hours after inoculation (hai) only to the treatment with basidiocarp extract. The O2.- was detected mainly to the treatment with mycelium extract at 48 hai. All the treatments showed reaction for H2O2 and O2.- in epidermal and mesophyllic cells at 192 hai, probably due the infection development. These results suggest that *P. sanguineus* extracts promote oxidative burst in bean plants, in early infection process, reducing anthracnose severity [18].

Thus, this work aimed to investigate the potential of *P. sanguineus* for controlling angular leaf spot in common bean, evaluating the *in vitro* antimicrobial activity against *P. griseola* and the induction of resistant enzymes as peroxidase, polyphenoloxidase and -1,3 glucanase, as well as the influence on physiological mechanisms related to the energy supply, as the protein content and chlorophyll.

## **2. Materials and methods**

**Pathogen isolate:** *Pseudocercospora griseola* was obtained from bean plants naturally infected, and cultivated in tomato juice (200 mL of tomato juice, 15 g of agar, 4,5 g of CaCO3 and 800 mL of distillated water) for 14 days at 24 °C and dark [19].

**Aqueous extract of** *Pycnoporus sanguineus* **fruiting body:** This process was carried out as methodologies [7] and [13]*.* Fruiting bodies or basidiocarps of *P. sanguineus* were collected in western Paraná State, Brazil, and identified according [10]. To obtain the aqueous extract, dehydrated powder from basidiocarps was suspended into distilled water (14 mL g-1) and, after 24 h incubation at 4ºC, the suspension was filtered through a common filter paper (8 g cm-2) and centrifuged at 20,000 *g* for 25 min. The supernatant obtained, after this procedure, was considered as the crude aqueous extract.

**Inhibition of conidia germination:** This assay was done in microscopic slide covered with a thin layer of agar-water 1% (700 L per slide) [20]. Aliquots of 40 L of aqueous extracts in concentrations 0, 1, 5, 10, 15 and 20%, sterilized in autoclave or filtrated in nitrocellulose membrane (0.45 m of pore diameter) and aliquots of 40 L of conidia suspension of *P. griseola* (1x104 conidia mL-1) obtained of a culture with 14 days old, were distributed in the surface of the slide, which were incubated in moist chamber under dark at 24 °C [21]. As control treatments were utilized fungicide (azoxystrobin: 40 mg L-1) and acibenzolar-Smethyl (ASM: 75 mg L-1). The percentage of the germination was determined after 24 hours with the addition of 40 L of lactophenol cotton-blue in each slide to paralyze the conidia germination.

154 Plant Science

*P. sanguineus* depending of its crude or aqueous extract and not of its individual

Previous works had shown the potential of *P. sanguineus* components for controlling plant diseases. Aqueous extracts, obtained from liquid medium-culture filtrate (MCF) [15] and from mycelium (AEM) [16] of *P. sanguineus*, were capable to reduce in 82% and 49% to MCF, and in 93% and 50% to AEM, in greenhouse and field conditions, respectively, the severity of angular leaf spot in bean plants. However, the effect of *P. sanguineus* fruiting bodies in that pathosystem was not investigated. Against plant pathogenic bacteria, fruiting body extracts from *P. sanguineus* were efficient for the control of common bacterial blight in bean, caused by *Xanthomonas axonopodis* pv. *phaseoli* (Smith – Vauterin, Hoste, Kersters & Swings) which can occur either by direct antimicrobial activity and by resistance induction involving

In another experiment, the *in situ* detection of reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2) and superoxide (O2.-), was searched in bean plants treated with aqueous extracts of the mycelium (AEM) and basidiocarps or fruiting bodies (AEB) of *P. sanguineus* and inoculated after three days with *Colletotrichum lindemuthianum* ((Sacc. & Magn.) Scrib.). It was possible to detect H2O2 at 48 hours after inoculation (hai) only to the treatment with basidiocarp extract. The O2.- was detected mainly to the treatment with mycelium extract at 48 hai. All the treatments showed reaction for H2O2 and O2.- in epidermal and mesophyllic cells at 192 hai, probably due the infection development. These results suggest that *P. sanguineus* extracts promote oxidative burst in bean plants, in early

Thus, this work aimed to investigate the potential of *P. sanguineus* for controlling angular leaf spot in common bean, evaluating the *in vitro* antimicrobial activity against *P. griseola* and the induction of resistant enzymes as peroxidase, polyphenoloxidase and -1,3 glucanase, as well as the influence on physiological mechanisms related to the energy

**Pathogen isolate:** *Pseudocercospora griseola* was obtained from bean plants naturally infected, and cultivated in tomato juice (200 mL of tomato juice, 15 g of agar, 4,5 g of CaCO3 and 800

**Aqueous extract of** *Pycnoporus sanguineus* **fruiting body:** This process was carried out as methodologies [7] and [13]*.* Fruiting bodies or basidiocarps of *P. sanguineus* were collected in western Paraná State, Brazil, and identified according [10]. To obtain the aqueous extract, dehydrated powder from basidiocarps was suspended into distilled water (14 mL g-1) and, after 24 h incubation at 4ºC, the suspension was filtered through a common filter paper (8 g cm-2) and centrifuged at 20,000 *g* for 25 min. The supernatant obtained, after this procedure,

compounds, like cinnabarin, or extracts obtained from organic solvents.

the activation of some pathogenesis-related proteins [17].

infection process, reducing anthracnose severity [18].

supply, as the protein content and chlorophyll.

was considered as the crude aqueous extract.

mL of distillated water) for 14 days at 24 °C and dark [19].

**2. Materials and methods** 

**Inhibition of the mycelial growth and sporulation of** *P. griseola***:** The extracts of basidiocarp of *P. sanguineus* were incorporated in concentrations of 0, 1, 5, 10, 15 and 20%, in tomato juice culture medium. The extracts were sterilized in autoclave and also by filtration in nitrocellulose membrane (0.45 m of diameter of pore) [22]. As control treatments were utilized fungicide (azoxystrobin: 40 mg L-1) and acibenzolar-S-methyl (ASM: 75 mg L-1). For transferring *P. griseola* to Petri dishes, 100 µL of spore suspension (1x104 conidia mL-1) were added to the medium and homogenized with Drygalski loop. The Petri dishes were sealed with plastic film and maintained at 24 °C and dark. Were evaluated the diameter and the number of colonies 14 days after the beginning of the experiment. At the end of the assay of mycelial growth inhibition, it was evaluated the sporulation of the fungus. For this, was prepared a suspension of conidia by the addition of 10 mL of distillated water per plate and determined the number of conidian per mL in Neubauer chamber.

**Experiment in greenhouse:** Two plants of common bean (cultivar IAPAR 81 – Carioca) were cultivated in plastic pots containing 5 L of a mixture of sterilized soil and sand (proportion 2:1). To resistance induction assay, were used aqueous extract of *P. sanguineus* basidiocarp at concentration of 10% and 20%. As control treatments were used water, fungicide (azoxystrobin - 40 mg L-1) and acibenzolar-S-methyl (75 mg L-1). The extracts and the control treatments were sprayed in the 3rd leaf (vegetative stage V4) (3 mL per leaf).

**Field experiment:** The experiment consisted in three randomized blocks, with five plots per block. Each plot consisted of three lines of 3 m of length, spaced 0.5 m between them, with 10 plants (cultivar IAPAR 81 – Carioca) per meter. The central line, discounting 0.5 m from the anterior and posterior borders, was considerate as useful area for evaluation. For the assay of resistance induction, were sprayed aqueous extracts of *P. sanguineus* basidiocarp at concentrations of 10% and 20%, and as control treatments were used water, fungicide (azoxystrobin: 40 mg L-1) and the acibenzolar-S-methyl (ASM: 75 mg L-1). The extracts (5 mL per plant) were applied twice, the first one in vegetative stage (V3) and the second in reproductive stage (R3).

**Pathogen inoculation:** The conidia suspension of *P. griseola* was prepared in water with Tween 20 (one drop 500 mL-1), and the concentration adjusted to 4x104 conidian mL-1. The inoculation in the greenhouse was done three days after the application of extracts and control treatments, in the 3rd treated leaf, as well as in the 4th non-treated leaf (vegetative

stage V4), to verify a putative systemic resistance induction. After the inoculation, the plants were maintained in humidity chambers and dark at 24 °C during 48 hours and, later, maintained in greenhouse, according to methodology used by [19]. In the field, two inoculations were done, the first in vegetative stage (V3) and the second in reproductive stage (R3), both three days after the application of extracts and control treatments.

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 157

of acetone 80%, during 7 days in the dark at 25 °C. After this time was determined the absorbance at 663 nm and 645 nm for chlorophyll *a* and *b,* respectively. The concentration of chlorophyll *a* was obtained by the equation (0.0127A663) – (0.00269A645) and of chlorophyll *b* by the equation (0.0029A645) – (0.00468A663). The total chlorophyll content was obtained by adding the results of chlorophyll *a* and *b*. The values were expressed in mg g of fresh mass-1. **Statistical analysis:** The experiments were arranged in randomized blocks, with five treatments. The analyzes of variance (ANOVA) was done using the statistical program JMP (Statistical Analysis System SAS Institute Inc. USA, 1989 – 2000 version 4.0.0.), and the

There was no significant effect of the concentrations of *P. sanguineus* basidiocarp extract on the spores germination, mycelial growth and sporulation of *P. griseola* (data not shown), indicating the absence of direct antimicrobial activity of these extracts on the pathogen.

However, in the greenhouse and field experiments, the area under disease progress curve (AUDPC) of angular leaf spot showed that the plants treated with *P. sanguineus* extract at 10% and 20% differ from the water-treatment, with reduction of 42% and 54% in the 3rd leaf, respectively. In the 4th leaf was observed reduction of 69% in the AUDPC for the plants treated with basidiocarp extract at 20%, not differing from ASM and fungicide control

In the lower middle canopy, there was no statistical difference in AUDPC for basidiocarp extract when compared to water and ASM control treatments. The difference was observed just to fungicide treatment, which presented the better protection against angular leaf spot. To the upper middle canopy, was verified a reduction of 64% in the AUDPC for the treatment with basidiocarp extract at 20%, better then ASM, which is a commercial resistance inducers product. So, these results indicate the great potential of *P. sanguineus*

The biochemistry analysis reveled induction of peroxidase activity due the treatment with basidiocarp extract on the 3rd treated leaf, as well as in the 4th non-treated and inoculated leaf, demonstrating the systemic induction effect of *P. sanguineus* (Figure 1). In the 3rd leaf the basidiocarp extract at 10% reduced the activity of the enzyme when compared to the water-treatment and fungicide three days after the inoculation (DAI). To five DAI differed from ASM and fungicide, and to seven DAI from ASM. The basidiocarp at 20% presented higher activity of peroxidase at four DAI, and inhibition to three, five and seven DAI, when compared to ASM, ASM and fungicide and ASM, respectively. The 4th non-treated leaf

The polyphenoloxidase activity was influenced by treatments with extracts of *P. sanguineus*, in the 3rd leaf treated, as well as in the 4th non-treated and inoculated leaf, demonstrating systemic effect (Figure 2). In the treated leaf, the extract of basidiocarp at 10% reduced the enzyme activity when compared to the water and ASM at three DAI. To four DAI the

extracts for the control of *P. griseola* in common bean, with local and systemic effects.

average compared by the Dunnett's test in level of 5% of probability.

treatments (Table 1), indicating systemic resistance induction.

showed the same pattern of 3rd leaf for peroxidase activity.

**3. Results** 

**Severity evaluation:** The severity of the angular leaf spot in the greenhouse was evaluated in the 3rd and 4th leaves at 8, 12, 16, 20 and 24 days after the inoculation, using diagrammatic scale prepared by [23]. In the field, the evaluations started when the first symptoms of disease appeared (seven days after the inoculation), and were obtained five evaluations on the lower middle canopy of the plant. In the second application of extracts and control treatments, the severity was evaluated as the same way that was done in the first application, but only evaluating the upper middle canopy. With the severity data was calculated the area under the disease progress curve (AUDPC) of angular leaf spot as in reference [24].

**Biochemical analysis:** Leaf disc with 3.46 cm2 (three disc per sample) were collected at 48, 72, 96, and 120 hours after the inoculation (hai) and also after the symptoms appearance (144 hai). Each collected sample was immediately wrapped in aluminum foil and freeze at -20 °C. Samples were collected from the 3rd treated and inoculated leaf, as well as from the 4th nontreated but inoculated leaf, from the same plant [25].

**Obtaining the protein extracts:** the samples of leaves were mechanically homogenized in 2 mL of extraction buffer sodium phosphate 0.01 M (pH 6.0), in a porcelain mortar. The homogenate was centrifuged at 6.500 *g* during 10 min at 4 °C. The supernatant was considerate the enzymatic extract, for later determination of peroxidase, polyphenoloxidase and -1,3-glucanase activities and protein content [25].

**Peroxidase activity:** the peroxidase activity was determined at 30 °C, by spectrophotometer at 470 nm during 2.15 min [26]. The peroxidase activity was expressed in absorbance min-1 g of fresh mass-1.

**Polyphenoloxidase activity:** the polyphenoloxidase activity was determined according the methodology in reference [27]. The results were expressed in absorbance min-1 g of fresh mass-1.

**-1,3 glucanase activity:** the enzyme activity was evaluated according to [19]. The reaction was determined by colorimetric quantification of glucose released from laminarin, using *p*hydroxybenzhydrazide. The results were expressed in g of glucose min-1 g of fresh mass-1.

**The protein content:** the total protein content was evaluated as [28]. The concentration of proteins, expressed in equivalent of bovine serum albumin (BSA) in one mL of sample (g protein mL-1), was determined utilizing standard curve of concentrations of BSA, varying of 0 to 20 g mL.

**The chlorophyll content:** for the quantification of chlorophyll was utilized an adapted methodology [29]. The samples of plant tissue (0.1 g) were packed in glass tube with 10 mL of acetone 80%, during 7 days in the dark at 25 °C. After this time was determined the absorbance at 663 nm and 645 nm for chlorophyll *a* and *b,* respectively. The concentration of chlorophyll *a* was obtained by the equation (0.0127A663) – (0.00269A645) and of chlorophyll *b* by the equation (0.0029A645) – (0.00468A663). The total chlorophyll content was obtained by adding the results of chlorophyll *a* and *b*. The values were expressed in mg g of fresh mass-1.

**Statistical analysis:** The experiments were arranged in randomized blocks, with five treatments. The analyzes of variance (ANOVA) was done using the statistical program JMP (Statistical Analysis System SAS Institute Inc. USA, 1989 – 2000 version 4.0.0.), and the average compared by the Dunnett's test in level of 5% of probability.

## **3. Results**

156 Plant Science

reference [24].

fresh mass-1.

0 to 20 g mL.

mass-1.

stage V4), to verify a putative systemic resistance induction. After the inoculation, the plants were maintained in humidity chambers and dark at 24 °C during 48 hours and, later, maintained in greenhouse, according to methodology used by [19]. In the field, two inoculations were done, the first in vegetative stage (V3) and the second in reproductive

**Severity evaluation:** The severity of the angular leaf spot in the greenhouse was evaluated in the 3rd and 4th leaves at 8, 12, 16, 20 and 24 days after the inoculation, using diagrammatic scale prepared by [23]. In the field, the evaluations started when the first symptoms of disease appeared (seven days after the inoculation), and were obtained five evaluations on the lower middle canopy of the plant. In the second application of extracts and control treatments, the severity was evaluated as the same way that was done in the first application, but only evaluating the upper middle canopy. With the severity data was calculated the area under the disease progress curve (AUDPC) of angular leaf spot as in

**Biochemical analysis:** Leaf disc with 3.46 cm2 (three disc per sample) were collected at 48, 72, 96, and 120 hours after the inoculation (hai) and also after the symptoms appearance (144 hai). Each collected sample was immediately wrapped in aluminum foil and freeze at -20 °C. Samples were collected from the 3rd treated and inoculated leaf, as well as from the 4th non-

**Obtaining the protein extracts:** the samples of leaves were mechanically homogenized in 2 mL of extraction buffer sodium phosphate 0.01 M (pH 6.0), in a porcelain mortar. The homogenate was centrifuged at 6.500 *g* during 10 min at 4 °C. The supernatant was considerate the enzymatic extract, for later determination of peroxidase, polyphenoloxidase

**Peroxidase activity:** the peroxidase activity was determined at 30 °C, by spectrophotometer at 470 nm during 2.15 min [26]. The peroxidase activity was expressed in absorbance min-1 g of

**Polyphenoloxidase activity:** the polyphenoloxidase activity was determined according the methodology in reference [27]. The results were expressed in absorbance min-1 g of fresh

**-1,3 glucanase activity:** the enzyme activity was evaluated according to [19]. The reaction was determined by colorimetric quantification of glucose released from laminarin, using *p*hydroxybenzhydrazide. The results were expressed in g of glucose min-1 g of fresh mass-1. **The protein content:** the total protein content was evaluated as [28]. The concentration of proteins, expressed in equivalent of bovine serum albumin (BSA) in one mL of sample (g protein mL-1), was determined utilizing standard curve of concentrations of BSA, varying of

**The chlorophyll content:** for the quantification of chlorophyll was utilized an adapted methodology [29]. The samples of plant tissue (0.1 g) were packed in glass tube with 10 mL

treated but inoculated leaf, from the same plant [25].

and -1,3-glucanase activities and protein content [25].

stage (R3), both three days after the application of extracts and control treatments.

There was no significant effect of the concentrations of *P. sanguineus* basidiocarp extract on the spores germination, mycelial growth and sporulation of *P. griseola* (data not shown), indicating the absence of direct antimicrobial activity of these extracts on the pathogen.

However, in the greenhouse and field experiments, the area under disease progress curve (AUDPC) of angular leaf spot showed that the plants treated with *P. sanguineus* extract at 10% and 20% differ from the water-treatment, with reduction of 42% and 54% in the 3rd leaf, respectively. In the 4th leaf was observed reduction of 69% in the AUDPC for the plants treated with basidiocarp extract at 20%, not differing from ASM and fungicide control treatments (Table 1), indicating systemic resistance induction.

In the lower middle canopy, there was no statistical difference in AUDPC for basidiocarp extract when compared to water and ASM control treatments. The difference was observed just to fungicide treatment, which presented the better protection against angular leaf spot. To the upper middle canopy, was verified a reduction of 64% in the AUDPC for the treatment with basidiocarp extract at 20%, better then ASM, which is a commercial resistance inducers product. So, these results indicate the great potential of *P. sanguineus* extracts for the control of *P. griseola* in common bean, with local and systemic effects.

The biochemistry analysis reveled induction of peroxidase activity due the treatment with basidiocarp extract on the 3rd treated leaf, as well as in the 4th non-treated and inoculated leaf, demonstrating the systemic induction effect of *P. sanguineus* (Figure 1). In the 3rd leaf the basidiocarp extract at 10% reduced the activity of the enzyme when compared to the water-treatment and fungicide three days after the inoculation (DAI). To five DAI differed from ASM and fungicide, and to seven DAI from ASM. The basidiocarp at 20% presented higher activity of peroxidase at four DAI, and inhibition to three, five and seven DAI, when compared to ASM, ASM and fungicide and ASM, respectively. The 4th non-treated leaf showed the same pattern of 3rd leaf for peroxidase activity.

The polyphenoloxidase activity was influenced by treatments with extracts of *P. sanguineus*, in the 3rd leaf treated, as well as in the 4th non-treated and inoculated leaf, demonstrating systemic effect (Figure 2). In the treated leaf, the extract of basidiocarp at 10% reduced the enzyme activity when compared to the water and ASM at three DAI. To four DAI the

enzyme activity was stimulated when compared to water and fungicide, and to five DAI was smaller than ASM. The basidiocarp at 20% showed increase in activity of polyphenoloxidase in relation to fungicide at three DAI, at four and five DAI, when compared to the three control treatments and at seven DAI in relation to fungicide. The 4th non-treated leaf presented similar effects to the 3rd leaf on the activity of polyphenoloxidase.

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 159

**Figure 2.** Polyphenoloxidase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide

PF: fresh mass.

3rd leaf on the activity of -1,3-glucanase.

(azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3).

The activity of -1,3-glucanase was influenced by the treatments with extract of *P. sanguineus*, in the 3rd leaf, as well as in the 4th non-treated and inoculated leaf (Figure 3). In the treated leaf, the extracts of basidiocarp at 10% and 20% reduced the enzyme activity in relation to control treatments at five DAI, and at seven DAI for the extract at 20% when compared to water and fungicide. In the 4th non-treated leaf there was similar effect to the

**Figure 3.** -1,3-glucanase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test,

P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.


Averages followed by a bold number differ statistically (Dunnett's test, P≤0.05) of the control treatments water (1), ASM (2) or fungicide (3);

\*3rd leaf: treated and inoculated; 4th leaf: non-treated and inoculated from same plant;

\*\*ASM: acibenzolar-S-methyl (75mg L-1);

\*\*\*Fungicide: azoxystrobin (40 mg L-1);

\*\*\*\*LMC and UMC: Lower middle canopy and upper middle canopy of the plant, respectively.

**Table 1.** Area under disease progress curve (AUDPC) of angular leaf spot in common bean after the application of aqueous extracts of *P. sanguineus* basidiocarp, in greenhouse and field conditions.

**Figure 1.** Peroxidase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

ASM (2) or fungicide (3);

\*\*ASM: acibenzolar-S-methyl (75mg L-1); \*\*\*Fungicide: azoxystrobin (40 mg L-1);

enzyme activity was stimulated when compared to water and fungicide, and to five DAI was smaller than ASM. The basidiocarp at 20% showed increase in activity of polyphenoloxidase in relation to fungicide at three DAI, at four and five DAI, when compared to the three control treatments and at seven DAI in relation to fungicide. The 4th non-treated leaf presented similar effects to the 3rd leaf on the activity of polyphenoloxidase.

 **3rd Leaf\* 4th Leaf\* LMC\*\*\*\* UMC\*\*\*\*** 

**Treatments Greenhouse Field**

\*3rd leaf: treated and inoculated; 4th leaf: non-treated and inoculated from same plant;

\*\*\*\*LMC and UMC: Lower middle canopy and upper middle canopy of the plant, respectively.

Basidiocarp 10% 24.5**1,2,3** 5.3 51.7**3** 54.2**1,3**

Basidiocarp 20% 19.5**1,2,3** 2.7**1** 49.9**3** 32.3**1,2,3** Water 41.9 8.8 59.4 89.0 ASM\*\* 2.3 5.2 41.9 67.7 Fungicide\*\*\* 6.7 2.4 6.3 16.1 C.V. (%) 31.4 7.3 47.5 53.9 Averages followed by a bold number differ statistically (Dunnett's test, P≤0.05) of the control treatments water (1),

**Table 1.** Area under disease progress curve (AUDPC) of angular leaf spot in common bean after the application of aqueous extracts of *P. sanguineus* basidiocarp, in greenhouse and field conditions.

**Figure 1.** Peroxidase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05)

from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

**AUDPC**

**Figure 2.** Polyphenoloxidase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

The activity of -1,3-glucanase was influenced by the treatments with extract of *P. sanguineus*, in the 3rd leaf, as well as in the 4th non-treated and inoculated leaf (Figure 3). In the treated leaf, the extracts of basidiocarp at 10% and 20% reduced the enzyme activity in relation to control treatments at five DAI, and at seven DAI for the extract at 20% when compared to water and fungicide. In the 4th non-treated leaf there was similar effect to the 3rd leaf on the activity of -1,3-glucanase.

**Figure 3.** -1,3-glucanase activity in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

The content of protein was significantly altered, both in the leaf treated with *P. sanguineus* extracts, as well as in the non-treated leaf (Figure 4). The effect of basidiocarp extract at 10% was significant at four DAI, stimulating the content of protein in relation to water and to fungicide. At five and seven DAI the effect was superior to water and to ASM. The basidiocarp at 20% was superior to water and to fungicide (three and four DAI) and at five and seven DAI was bigger than the three control treatments. This effect of *P. sanguineus*  extracts on the protein content was similar for the 4th non-treated leaf.

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 161

time-dependent when it is considered the time interval between induction and pathogen

**Figure 5.** Chlorophylls *a, b* and total content in bean plants inoculated with *Pseudocercospora griseola* tree

The activities of peroxidase, polyphenoloxidase and -1,3 glucanase, and the content of total proteins and chlorophylls were altered in plants treated with *P. sanguineus* extract. Changes in the activities of peroxidase have being frequently correlated to the answer of resistance or susceptibility in different pathosystems. The peroxidase is responsible for the remove of atoms

(azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3).

days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide

inoculation.

PF: fresh mass.

**Figure 4.** Protein content in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

The content of chlorophylls *a, b* and total in common bean treated with aqueous extracts of *P. sanguineus* basidiocarp and challenged with *P. griseola* was altered significantly, with increments in the level of pigments (Figure 5). It was verified a similar behavior in the chlorophyll content in the 4th non-treated leaf, emphasizing the systemic effects of *P. sanguineus* on the content of total chlorophyll in common bean.

### **4. Discussion**

The basidiocarp extracts did not present direct antimicrobial activity on *P. griseola*. This is a satisfactory result, since for a product be considerate a resistance inductor, this should not show antimicrobial activity *in vitro* or *in vivo* assays [30].

In greenhouse and field it was observed reduction of AUDPC in plants treated with the extracts of *P. sanguineus*. In [13] the authors obtained similar results with aqueous extracts of the same basidiocarp, reducing the severity of anthracnose in common bean in a systemic way. In reference [31] was demonstrated partial reduction in the severity of anthracnose in cucumber leaf pre-treated with the fruiting bodies extracts of *Lentinula edodes* and *Agaricus blazei,* in a systemic way. The protection effect was dose-dependent and, in a minor degree,

time-dependent when it is considered the time interval between induction and pathogen inoculation.

160 Plant Science

The content of protein was significantly altered, both in the leaf treated with *P. sanguineus* extracts, as well as in the non-treated leaf (Figure 4). The effect of basidiocarp extract at 10% was significant at four DAI, stimulating the content of protein in relation to water and to fungicide. At five and seven DAI the effect was superior to water and to ASM. The basidiocarp at 20% was superior to water and to fungicide (three and four DAI) and at five and seven DAI was bigger than the three control treatments. This effect of *P. sanguineus* 

**Figure 4.** Protein content in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05)

The content of chlorophylls *a, b* and total in common bean treated with aqueous extracts of *P. sanguineus* basidiocarp and challenged with *P. griseola* was altered significantly, with increments in the level of pigments (Figure 5). It was verified a similar behavior in the chlorophyll content in the 4th non-treated leaf, emphasizing the systemic effects of *P.* 

The basidiocarp extracts did not present direct antimicrobial activity on *P. griseola*. This is a satisfactory result, since for a product be considerate a resistance inductor, this should not

In greenhouse and field it was observed reduction of AUDPC in plants treated with the extracts of *P. sanguineus*. In [13] the authors obtained similar results with aqueous extracts of the same basidiocarp, reducing the severity of anthracnose in common bean in a systemic way. In reference [31] was demonstrated partial reduction in the severity of anthracnose in cucumber leaf pre-treated with the fruiting bodies extracts of *Lentinula edodes* and *Agaricus blazei,* in a systemic way. The protection effect was dose-dependent and, in a minor degree,

extracts on the protein content was similar for the 4th non-treated leaf.

from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

*sanguineus* on the content of total chlorophyll in common bean.

show antimicrobial activity *in vitro* or *in vivo* assays [30].

**4. Discussion** 

**Figure 5.** Chlorophylls *a, b* and total content in bean plants inoculated with *Pseudocercospora griseola* tree days after the application of water (), acibenzolar-S-methyl (ASM 75mg L-1) (), fungicide (azoxystrobin 40mg L-1) () and the aqueous extracts of basidiocarp of *Pycnoporus sanguineus* at 10% and 20% ( and ). **A** and **B** represent the 3rd treated and inoculated leaf and 4th non-treated and inoculated leaf, respectively. Bars indicate an average ± standard error. Average followed by \* differ statistically (Dunnett's test, P≤0.05) from the control treatments water (1), ASM (2) or fungicide (3). PF: fresh mass.

The activities of peroxidase, polyphenoloxidase and -1,3 glucanase, and the content of total proteins and chlorophylls were altered in plants treated with *P. sanguineus* extract. Changes in the activities of peroxidase have being frequently correlated to the answer of resistance or susceptibility in different pathosystems. The peroxidase is responsible for the remove of atoms

of hydrogen of the hydroxyl cinnamic alcohols groups, whose radical polymerize to form the lignin. This polymer, together with cellulose and other polysaccharides occurring in the cell wall of the superior plants, works as a physical barrier to the pathogen penetration [4].

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 163

Protein synthesis could be related with the increase of the demand for substrates, necessary to the production of plant defense mechanisms induced by *P. sanguineus* treatment. Among the proteins, there are the pathogenesis related proteins (PR-proteins) which are induced in plant tissues due to inoculation with pathogens/microorganisms, systemically or local, as well as with treatments with chemical agents [38]. The activation of protein synthesis leads to a phase of plant resistance [37]. In [32] was verified reduction in protein content of bean plants when treated with *Bacillus cereus*, contrary to the treatment with ASM, demonstrating

The chlorophyll content *a, b* and total in bean plants treated with *P. sanguineus* extract and challenged with *P. griseola* was altered significantly, with increase in the levels of these pigments. These results suggest the need to generate energy for the synthesis of compounds involved in plant defense, considering that the chlorophyll molecules *a* and *b* constitute the two pigment systems responsible for the absorption and transference of the radiant energy [39]. The energy generated by the process of photosynthesis can, at a given time, be directed to the production of secondary metabolic compounds, as for example, in case of attack by pathogens [37]. An increase was observed in the levels of chlorophyll in regions infected with *U. appendiculatus*, which occurs both in the common bean cultivar moderately

In this work can be concluded that the extract from *P.* s*anguineus* basidiocarp reduce the severity of the angular leaf spot in common bean, by increasing the activity of defense enzymes peroxidase, polyphenoloxidase and -1,3-glucanase, local and systemically. Additionally, physiological changes in the content of protein and chlorophylls were verified, probably due the apparatus energy synthesis required for plant defense mechanism involved in the reduction of this disease. In this way, the use of extracts from *P. sanguineus*

, Odair José Kuhn, Lindomar Assi and Cristiane Cláudia Meinerz

fruiting bodies for the control of plant diseases in organic growth shows promising.

*Western Paraná State University – UNIOESTE, Marechal Cândido Rondon, Paraná, Brazil* 

specificity in the physiological response of this host to the elicitor treatment.

susceptible as in cultivar highly susceptible to pathogen [25].

*Assis Gurgacz Foundation – FAG, Cascavel, Paraná, Brazil* 

*Maringá State University – UEM, Maringá, Paraná, Brazil* 

*Paraná Federal University – UFPR, Palotina, Paraná, Brazil* 

**5. Conclusions** 

**Author details** 

José Renato Stangarlin\*

Clair Aparecida Viecelli

Roberto Luis Portz

Corresponding Author

 \*

Kátia Regina Freitas Schwan-Estrada

In [13] was verified peroxidase induction, local and systemically, in bean plants treated with aqueous extract of *P. sanguineus* and challenged with *C. lindemuthianum*, agreeing with the results obtained in this work. In reference [14] the authors evaluated the effects of organic extracts of *P. sanguineus* basidiocarp, and verified that the dichloromethane extract for sorghum and soybean, and ethanolic extract for soybean, inhibit the activity of peroxidase, while the hexanic extract promotes the activity for sorghum and soybean. In another work [32], the peroxidase activity in the common bean was influenced, in a time-dependent way, by the number of inducer applications. The inducer ASM promoted increase in the enzyme activity in a more accentuated way and faster than the biotic inducer *Bacillus cereus*. In [33] it was not found significant increments in activity of this enzyme in the common bean treated with *P. sanguineus* extract and inoculated with *C. lindemuthianum.* In [32] was observed that the activity of polyphenoloxidase in common bean was not altered by the treatment with *Bacillus cereus* and ASM, while in [34] was verified induction in the activity of these enzyme in tomato leaves treated with essential oil of *Cymbopogon citratus* and inoculated with *Alternaria solani.*

In the resistance induction, the increment of -1,3-glucanase is related with the plant defense. This enzyme hydrolyzes -1,3-glucan, which, together chitin, is the main component of fungal cell wall [35]. In another pathosystem [33] was observed increase in specific activity of -1,3-glucanase in common bean treated with *P. sanguineus* extract and challenge with *C. lindemuthianum.* In the 1st leaf, treated and inoculated, the activity increased 28%, while in the 2nd leaf, non-treated, but inoculated, the culture filtrate at 5% and the mycelium extract at 10% increased in 331% and 1,057%, respectively, the enzyme activity.

Extracts from other basidiomycetes fruiting bodies have also induced the activity of -1,3 glucanase. It was verified an increase in this enzyme in passion fruit inoculated with *Xanthomonas campestris* pv. *passiflorae* and treated with extracts of *L. edodes* and *A. blazei* in concentration of 20% and 40% [36]. According [19], bean plants challenged with *P. griseola*  did not presented induction in the -1,3-glucanase activity, however, when challenged with *Uromyces appendiculatus*, was verified induction of this enzyme [25]. This behavior indicates the differential interaction among the elicitors treatment and pathogens challenging, in the activation of defense mechanisms in plants. The plant could invest in the production of compounds that normally would be produced in the presence of the pathogen, however, with greater efficiency when pre-disposed to an elicitor.

In this work, the protein content was significantly altered both in the leaf treated with the *P. sanguineus* extracts as in the non-treated leaf, however, there was a faster response on protein synthesis in the 4th leaf. This result could be related to the age of the leaves in the moment of treatments application and pathogen inoculation, since the 4th leaf has probably more physiological activity than 3rd one, optimizing the protein synthesis and plant resistance response [37].

Protein synthesis could be related with the increase of the demand for substrates, necessary to the production of plant defense mechanisms induced by *P. sanguineus* treatment. Among the proteins, there are the pathogenesis related proteins (PR-proteins) which are induced in plant tissues due to inoculation with pathogens/microorganisms, systemically or local, as well as with treatments with chemical agents [38]. The activation of protein synthesis leads to a phase of plant resistance [37]. In [32] was verified reduction in protein content of bean plants when treated with *Bacillus cereus*, contrary to the treatment with ASM, demonstrating specificity in the physiological response of this host to the elicitor treatment.

The chlorophyll content *a, b* and total in bean plants treated with *P. sanguineus* extract and challenged with *P. griseola* was altered significantly, with increase in the levels of these pigments. These results suggest the need to generate energy for the synthesis of compounds involved in plant defense, considering that the chlorophyll molecules *a* and *b* constitute the two pigment systems responsible for the absorption and transference of the radiant energy [39]. The energy generated by the process of photosynthesis can, at a given time, be directed to the production of secondary metabolic compounds, as for example, in case of attack by pathogens [37]. An increase was observed in the levels of chlorophyll in regions infected with *U. appendiculatus*, which occurs both in the common bean cultivar moderately susceptible as in cultivar highly susceptible to pathogen [25].

## **5. Conclusions**

162 Plant Science

of hydrogen of the hydroxyl cinnamic alcohols groups, whose radical polymerize to form the lignin. This polymer, together with cellulose and other polysaccharides occurring in the cell

In [13] was verified peroxidase induction, local and systemically, in bean plants treated with aqueous extract of *P. sanguineus* and challenged with *C. lindemuthianum*, agreeing with the results obtained in this work. In reference [14] the authors evaluated the effects of organic extracts of *P. sanguineus* basidiocarp, and verified that the dichloromethane extract for sorghum and soybean, and ethanolic extract for soybean, inhibit the activity of peroxidase, while the hexanic extract promotes the activity for sorghum and soybean. In another work [32], the peroxidase activity in the common bean was influenced, in a time-dependent way, by the number of inducer applications. The inducer ASM promoted increase in the enzyme activity in a more accentuated way and faster than the biotic inducer *Bacillus cereus*. In [33] it was not found significant increments in activity of this enzyme in the common bean treated with *P. sanguineus* extract and inoculated with *C. lindemuthianum.* In [32] was observed that the activity of polyphenoloxidase in common bean was not altered by the treatment with *Bacillus cereus* and ASM, while in [34] was verified induction in the activity of these enzyme in tomato leaves treated with essential oil of *Cymbopogon citratus* and inoculated with *Alternaria solani.*

In the resistance induction, the increment of -1,3-glucanase is related with the plant defense. This enzyme hydrolyzes -1,3-glucan, which, together chitin, is the main component of fungal cell wall [35]. In another pathosystem [33] was observed increase in specific activity of -1,3-glucanase in common bean treated with *P. sanguineus* extract and challenge with *C. lindemuthianum.* In the 1st leaf, treated and inoculated, the activity increased 28%, while in the 2nd leaf, non-treated, but inoculated, the culture filtrate at 5% and the mycelium extract at 10% increased in 331% and 1,057%, respectively, the enzyme activity.

Extracts from other basidiomycetes fruiting bodies have also induced the activity of -1,3 glucanase. It was verified an increase in this enzyme in passion fruit inoculated with *Xanthomonas campestris* pv. *passiflorae* and treated with extracts of *L. edodes* and *A. blazei* in concentration of 20% and 40% [36]. According [19], bean plants challenged with *P. griseola*  did not presented induction in the -1,3-glucanase activity, however, when challenged with *Uromyces appendiculatus*, was verified induction of this enzyme [25]. This behavior indicates the differential interaction among the elicitors treatment and pathogens challenging, in the activation of defense mechanisms in plants. The plant could invest in the production of compounds that normally would be produced in the presence of the pathogen, however,

In this work, the protein content was significantly altered both in the leaf treated with the *P. sanguineus* extracts as in the non-treated leaf, however, there was a faster response on protein synthesis in the 4th leaf. This result could be related to the age of the leaves in the moment of treatments application and pathogen inoculation, since the 4th leaf has probably more physiological activity than 3rd one, optimizing the protein synthesis and plant

with greater efficiency when pre-disposed to an elicitor.

resistance response [37].

wall of the superior plants, works as a physical barrier to the pathogen penetration [4].

In this work can be concluded that the extract from *P.* s*anguineus* basidiocarp reduce the severity of the angular leaf spot in common bean, by increasing the activity of defense enzymes peroxidase, polyphenoloxidase and -1,3-glucanase, local and systemically. Additionally, physiological changes in the content of protein and chlorophylls were verified, probably due the apparatus energy synthesis required for plant defense mechanism involved in the reduction of this disease. In this way, the use of extracts from *P. sanguineus* fruiting bodies for the control of plant diseases in organic growth shows promising.

## **Author details**

José Renato Stangarlin\* , Odair José Kuhn, Lindomar Assi and Cristiane Cláudia Meinerz *Western Paraná State University – UNIOESTE, Marechal Cândido Rondon, Paraná, Brazil* 

Clair Aparecida Viecelli *Assis Gurgacz Foundation – FAG, Cascavel, Paraná, Brazil* 

Kátia Regina Freitas Schwan-Estrada *Maringá State University – UEM, Maringá, Paraná, Brazil* 

Roberto Luis Portz *Paraná Federal University – UFPR, Palotina, Paraná, Brazil* 

<sup>\*</sup> Corresponding Author

## **Acknowledgement**

The authors thank the Araucaria Foundation and the FINEP for the financial support for the realization of this work. JRS and KRFSE thank to CNPq for the scholarship of research productivity.

Plant Defense Enzymes Activated in

Bean Plants by Aqueous Extract from *Pycnoporus sanguineus* Fruiting Body 165

[13] Assi L. Control of *Colletotrichum lindemuthianum* (Sacc. Et Magn.) Scrib, in common bean (*Phaseolus vulgaris* L.) by *Pycnoporus sanguineus* (L. ex. Fr.) extract. Master thesis.

[14] Peiter-Beninca C, Franzener G, Assi L, Iurkiv L, Eckstein B, Costa VC, Nogueira MA, Stangarlin JR, Schwan-Estrada KRF. Phytoalexin Induction and Peroxidase Activity in Sorghum and Soybean Treated with Basidiocarp Extracts of *Pycnoporus sanguineus*.

[15] Viecelli CA, Stangarlin JR, Kuhn OJ, Schwan-Estrada KRF. Induction of Resistance in Beans against *Pseudocercospora griseola* by Culture Filtrates of *Pycnoporus sanguineus*.

[16] Viecelli CA, Stangarlin JR, Kuhn OJ, Schwan-Estrada KRF. Resistance Induction in Bean Plants against Angular Leaf Spot by Extracts from *Pycnoporus sanguineus* Mycelium.

[17] Toillier SL, Iurkiv L, Meienrz CC, Baldo M, Viecelli CA, Kuhn OJ, Schwan-Estrada KRF, Stangarlin JR. Control of Bacterial Blight (*Xanthomonas axonopodis* pv. *phaseoli*) and Biochemical Analyses of Bean Resistance Treated with *Pycnoporus sanguineus* Extracts.

[18] Baldo M, Stangarlin JR, Franzener G, Assi L, Kuhn OJ, Schwan-Estrada KRF. *In situ* Detection of Reactive Oxygen Species in Bean Treated with *Pycnoporus sanguineus* Extracts and Inoculated with *Colletotrichum lindemuthianum*. Summa Phytopathologica

[19] Stangarlin JR, Pascholati SF, Labate CA. Effect of *Phaeoisariopsis griseola* on Ribulose-1,5 bisphosphate Carboxylase-oxygenase, Chlorophyllase, β-1,3 glucanase and Chitinase Activities in *Phaseolus vulgaris* cultivars*.* Fitopatologia Brasileira 2000;25(1): 59-66. [20] Stangarlin JR, Schwan-Estrada KRF, Cruz MES, Nozaki MH. Medicinal Plants and Alternative Control of Phytopathogens. Biotecnologia Ciência & Desenvolvimento

[21] Fiori ACG, Schwan-Estrada KRF, Stangarlin JR, Vida JB, Scapim CA, Cruz MES. Antifungal Activity of Leaf Extracts and Essential Oils of some Medicinal Plants against

[22] Stangarlin JR, Franzener G, Schwan-Estrada KRF, Cruz MES. Estratégias de Seleção e Uso de Extratos de Plantas no Controle Microbiano *in vitro.* In: Scherwinski-Pereira JE (ed.) Contaminações microbianas na cultura de células, tecidos e órgãos de plantas.

[23] Godoy CV, Carneiro SMTPG, Iamauti MT, Dalla Pria M, Amorim L, Berger RD, Bergamin Filho A. Diagrammatic Scales for Bean Diseases: Development and Validation. Zeitschrift fur Planzenkrankheiten und Pflanzenschutz 1997;104(4): 336-345. [24] Shaner G, Finney RE. The Effect of Nitrogen Fertilization in the Expression of Slow

[25] Stangarlin JR, Pascholati SF. Activities of ribulose-1,5-bisphosphate Carboxylaseoxygenase (rubisco), chlorophyllase, -1,3 glucanase and Chitinase and Chlorophyll Content in Bean Cultivars (*Phaseolus vulgaris*) infected with *Uromyces appendiculatus*.

Mildewing Resistance in Knox Wheat. Phytopathology 1977;67: 1051-1056.

*Didymella bryoniae*. Journal of Phytopathology 2000;148: 483-487.

Western Parana State University; 2005.

Tropical Plant Pathology 2009;34: 87-96.

Summa Phytopathologica 2010;36: 73-80.

2011;37(4): 174-179.

1999;11: 16-21.

Brasília: Embrapa; 2010. p293-345.

Summa Phytopathologica 2000;26(1): 34-42.

Arquivos do Instituto Biológico 2010;77: 99-110.

Arquivos do Instituto Biológico 2008;75: 285-292.

#### **6. References**


[13] Assi L. Control of *Colletotrichum lindemuthianum* (Sacc. Et Magn.) Scrib, in common bean (*Phaseolus vulgaris* L.) by *Pycnoporus sanguineus* (L. ex. Fr.) extract. Master thesis. Western Parana State University; 2005.

164 Plant Science

productivity.

**6. References** 

**Acknowledgement** 

Paul: APS; 2005.

Badajoz: Formatex; 2011. p1033-1042.

The authors thank the Araucaria Foundation and the FINEP for the financial support for the realization of this work. JRS and KRFSE thank to CNPq for the scholarship of research

[1] Schwarrtz HF, Steadman JR, Hall R, Forster RL. Compendium of Bean Diseases. St.

[2] Stangarlin JR, Kuhn OJ, Schwan-Estrada KRF. Control of Plant Diseases by Plant

[3] Stangarlin JR, Kuhn OJ, Assi L, Schwan-Estrada KRF. Control of Plant Diseases using Extracts from Medicinal Plants and Fungi. In: Méndez-Vilas A. (ed.) Science against microbial pathogens: communicating current research and technological advances.

[4] Stangarlin JR, Kuhn OJ, Toledo MV, Portz RL, Schwan-Estrada KRF, Pascholati SF. Plant Defense against Pathogens. Scientia Agraria Paranaensis 2011;10(1): 18-46. [5] Schwan-Estrada KRF, Stangarlin JR. Extracts and Essential Oils of Medicinal Plants in the Resistance Induction against Plant Pathogens. In: Cavalcanti LS, Di Piero RM, Cia P, Pscholati SF, Resende MLV, Romeiro RS (ed.) Resistance induction in plants against

[6] Toledo MV, Stangarlin JR, Bonato CM. Homeopathy for the Control of Plant Pathogens. In: Méndez-Vilas A. (ed.) Science against microbial pathogens: communicating current

[7] Di Piero RM, Wulff NA, Pascholati SF. Partial Purification of Elicitors from *Lentinula edodes* Basidiocarps Protecting Cucumber Seedlings against *Colletotrichum lagenarium*.

[8] Di Piero RM, Novaes QS, Pascholati SF. Effect of *Agaricus brasiliensis* and *Lentinula edodes* Mushrooms on the Infection of Passionflower with Cowpea Aphid-borne Mosaic

[9] Fiori-Tutida ACG, Schwan-Estrada KRF, Stangarlin JR, Pascholati SF. Extracts of *Lentinula edodes* and *Agaricus blazei* on *Bipolaris sorokiniana* and *Puccinia recondita* f. sp.

[10] Nobles MK, Frew BP. Studies in Wood-inhabiting Hymenomycetes. The Genus

[11] Smânia A, Monache FD, Smânia EF, Gil ML, Benchetrit LC, Cruz FS. Antibacterial Activity of a Substance Produced by the Fungus *Pycnoporus sanguineus.* Journal of

[12] Smânia EFA, Smânia A, Loguercio-Leite C, Gil ML. Optimal Parameters for Cinnabarin Synthesis by *Pycnoporus sanguineus*. Journal of Chemical Technology and Biotechnology

research and technological advances. Badajoz: Formatex; 2011. p1063-1067.

Virus. Brazilian Archives of Biology and Technology 2010;53: 269-278.

Extracts. Revisão Anual de Patologia de Plantas 2008;16: 265-304.

pathogens and insects. Piracicaba: FEALQ; 2005. p125-138.

Brazilian Journal of Microbiology 2006;37(2): 169-174.

*tritici*, *in vitro*. Summa Phytopathologica 2007;33: 287-289.

Ethnopharmacology 1995;45: 177-81.

1997;70: 57-59.

*Pycnoporus* Karst. Canadian Journal of Botany 1962;40: 987-1016.


[26] Lusso MFG, Pascholati SF. Activity and Isoenzymatic Pattern of Soluble Peroxidases in Maize Tissues after Mechanical Injury or Fungal Inoculation. Summa Phytopathologica 1999;25: 244-249.

**Section 3** 

**Plant Biotechnology** 


**Plant Biotechnology** 

166 Plant Science

1999;25: 244-249.

1999;64: 351-359.

Biochemistry 1976;72: 248-254.

State University; 2008.

University; 2008.

283-332.

Pathology 2008;33(3): 241-244.

Plants. Plant Physiology 1993;101: 709-712.

[37] Larcher W. Plant Ecophysiology. São Carlos: RiMa; 2000.

[39] Taiz L, Zeiger E. Plant Physiology. Sunderland: Sinauer; 2006.

*vulgaris*. Plant Physiology 1949;24: 1-15.

*blazei*. Summa Phytophathologica 2004;30(2): 243-250.

Production Parameters. PhD thesis. ESALQ; 2007.

of Phytopathology 1997;35: 235-270.

[26] Lusso MFG, Pascholati SF. Activity and Isoenzymatic Pattern of Soluble Peroxidases in Maize Tissues after Mechanical Injury or Fungal Inoculation. Summa Phytopathologica

[27] Duangmal K, Apenten RKO. A Comparative Study of Polyphenoloxidases from Taro (*Colocasia esculenta*) and Potato (*Solanum tuberosum* var. Romano). Food Chemistry

[28] Bradford M.M. A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding. Analytical

[29] Arnon DI. Copper Enzymes in Isolated Chloroplasts. Polyphenoloxidase in *Beta* 

[30] Sticher L, Mauch-Mani B, Métraux J-P. Systemic Acquired Resistance. Annual Review

[31] Di Piero MR, Pascholati SF. Induced Resistance Cucumber Plants against *Colletotrichum lagenarium* by Application of Fruiting Body Extracts from *Lentinula edodes* and *Agaricus* 

[32] Kuhn OJ. Resistance Induced in bean Plants (*Phaseolus vulgaris*) by Acibenzolar-Smethyl and *Bacillus cereus*: Physiological and Biochemical Aspects, Growth and

[33] Baldo M. Histological and Biochemical Aspects of the Resistance Induction in Bean Plants and Antifungal Activity by *Pycnoporus sanguineus*. Master thesis. Western Paraná

[34] Itako, AT, Schwan-Estrada KRF, Tolentino Jr, Stangarlin JR, Cruz MES. Antifungal Activity and Protection of tomato Plants by Extracts of Medicinal Plants. Tropical Plant

[35] Cornelissen BJC, Melchers LS. Strategies for Control of Fungal Diseases with Transgenic

[36] Fiori-Suzuki CCL. Induction of Resistance in Yellow Passion Fruit (*Passiflora edulis* f. *flavicarpa*) by shiitake (*Lentinula edodes*) and *Agaricus blazei*. PhD thesis. Maringa State

[38] Guzzo SD. Pathogen Related Proteins. Revisão Anual de Patologias de Plantas 2003;11:

**Chapter 7** 

© 2012 López et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 López et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Small Non-Coding RNAs in Plant Immunity** 

During millions of years of co-evolution, plants have established sophisticated genetic mechanisms to protect their integrity against invading pathogens. Pathogens in turn have coped with such barriers to gain access to nutrients and proliferate inside the plant. The "zigzag model" illustrates in a simple way the different layers of innate immunity during interactions of plants with their pathogen [1]. This model describes two main immunity responses, the first one relies on plants' ability to recognize so-called microbial-associated molecular patterns (MAMPs), which are highly conserved structures and molecules in all kinds of pathogenic and nonpathogenic microorganisms. This response is known as MAMPtriggered immunity (MTI) and is efficient against non-adapted or non-host pathogens [2]. The best-studied MAMPs are the flagelline peptide, the elongation factor Tu protein (EF-Tu), chitin whis is a major component of fungal cell walls and lipopolysacharides (LPS). MAMPs perception depends on plant pathogen recognition receptors (PRRs), with FLS2 and EFR recognizing flagelline and EF-Tu, respectively. These two PRRs share a similar structural architecture formed by extracellular Leucine Rich Repeats (LRR) and a cytoplasmic kinase domain. CERK1, on the other hand, which is the Arabidopsis PRR involved in the recognition of chitine, contains three extracellular LysM domains and a cytoplasmic kinase domain. In response to MTI, pathogens developed strategies to overcome it by sending effector proteins inside plant cells. These effector proteins abolish MTI by either suppressing early recognition or interfering with down-stream signaling events [3]. A second layer of plant immunity known as effector-triggered immunity (ETI) relies on a more sophisticated mechanism to detect pathogens, based on the specific recognition of particular effector proteins by Resistance (R) proteins. In this case the effector proteins are named Avr factors. The Avr-R proteins interaction can be direct or mediated by another protein, referred to as pathogenicity target. In the later case the R protein guards the pathogenicity target and detects its modification caused by the effector protein [4]. The

Camilo López, Boris Szurek and Álvaro L. Perez-Quintero

**1.1. The "zigzag model" of plant-pathogen interactions** 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54086

**1. Introduction** 

## **Small Non-Coding RNAs in Plant Immunity**

Camilo López, Boris Szurek and Álvaro L. Perez-Quintero

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54086

#### **1. Introduction**

#### **1.1. The "zigzag model" of plant-pathogen interactions**

During millions of years of co-evolution, plants have established sophisticated genetic mechanisms to protect their integrity against invading pathogens. Pathogens in turn have coped with such barriers to gain access to nutrients and proliferate inside the plant. The "zigzag model" illustrates in a simple way the different layers of innate immunity during interactions of plants with their pathogen [1]. This model describes two main immunity responses, the first one relies on plants' ability to recognize so-called microbial-associated molecular patterns (MAMPs), which are highly conserved structures and molecules in all kinds of pathogenic and nonpathogenic microorganisms. This response is known as MAMPtriggered immunity (MTI) and is efficient against non-adapted or non-host pathogens [2]. The best-studied MAMPs are the flagelline peptide, the elongation factor Tu protein (EF-Tu), chitin whis is a major component of fungal cell walls and lipopolysacharides (LPS). MAMPs perception depends on plant pathogen recognition receptors (PRRs), with FLS2 and EFR recognizing flagelline and EF-Tu, respectively. These two PRRs share a similar structural architecture formed by extracellular Leucine Rich Repeats (LRR) and a cytoplasmic kinase domain. CERK1, on the other hand, which is the Arabidopsis PRR involved in the recognition of chitine, contains three extracellular LysM domains and a cytoplasmic kinase domain. In response to MTI, pathogens developed strategies to overcome it by sending effector proteins inside plant cells. These effector proteins abolish MTI by either suppressing early recognition or interfering with down-stream signaling events [3]. A second layer of plant immunity known as effector-triggered immunity (ETI) relies on a more sophisticated mechanism to detect pathogens, based on the specific recognition of particular effector proteins by Resistance (R) proteins. In this case the effector proteins are named Avr factors. The Avr-R proteins interaction can be direct or mediated by another protein, referred to as pathogenicity target. In the later case the R protein guards the pathogenicity target and detects its modification caused by the effector protein [4]. The

© 2012 López et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 López et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

largest group of R proteins includes a Nucleotide Binding Site (NBS) and a LRR domain. This group can be subdivided in two subclasses based on the presence or not of a Toll-Interleukin Related (TIR) domain in the N terminus. Upon the perception of pathogen's molecules by PRRs or R proteins, a signaling cascade including MAP kinases is activated, leading to a reprogramming in host's gene expression along with the activation of genes with antimicrobial function (PR, pathogenesis related) [2, 5]. In the last years a great effort in research has focused on understanding how gene expression is modified in response to pathogens, revealing some crucial factors for the interaction such as transcription factors, DNA regulatory elements and non-coding small RNAs.

Small Non-Coding RNAs in Plant Immunity 171

years we have learned a lot about how non coding RNAs are a crucial players in regulating

In eukaryotes, the endogenous regulation of gene expression is mostly dependent on the control of the RNA polymerase II by accessory proteins including activators, repressors and the mediator complex. Then, small non-coding RNAs (sncRNA) were discovered and found to be new key elements of gene expression regulation [9, 10]. sncRNAs are short molecules of typically 18 to 30 nt, involved in gene expression control, defense against other parasitic

The best-studied sncRNAs are microRNAs (miRNAs) and small interference RNAs (siRNAs). miRNAs derive from nuclear genes. A gene coding for a miRNA (*MIRNA*) is first transcribed by the RNA polymerase II to a primary miRNA (pri-miRNA), the size of which ranges from 100 nt to several kilobases (kb). A Dicer-like (DCL) protein DCL1 in Arabidopsis along with HYPONASTIC LEAVES1 (HYL1), process the pri-miRNA into a 70 to 400 nt long precursor miRNA (pre-miRNA). This pre-miRNA forms a characteristic hairpin-like structure. A subsequent processing step involving DCL slices the pre-miRNA to form a miRNA:miRNA\* duplex (21-22 nt). The duplex is then methylated by HEN1 and exported from the nucleus to the cytoplasm where it will join an AGO protein to form the silencing complex (RISC). Only the mature miRNA strand which is usually the one with less stable 5'-end pairing, is retained in the complex, while the passenger (miRNA\*) strand is degraded. The miRNA\* degradation process remain unknown, although some family of exoribonucleases encoded by the *SMALL RNA DEGRADING NUCLEASE* (*SDN*) genes degrades mature miRNAs which could also be involved in the miRNA\* degradation [11]. The miRNA retained in the RISC complex will then guide the silencing of complementary

In contrast, siRNAs originate from transgenes, viruses, transposons or other RNAs that form perfectly complementary double-stranded RNA precursors (dsRNAs). In particular, virusderived siRNAs also known as virus induced RNAs (vsiRNAs), have been extensively studied in plants. From a siRNA precursor, multiple siRNAs are generated and the silencing signal can be further amplified upon the generation of secondary siRNAs subsequently processed by RNA dependent RNA polymerases (RDRs), SILENCING DEFECTIVE3 (SDE3), NRPD1a and NRPD1b (largest subunits of Pol IVa and Pol IVb isoforms of RNA

Recently, various new types of ncRNAs have been described. Among them are the transacting siRNAs (ta-siRNAs) (21-22nt) which combine both the siRNA and miRNA pathways since they originate from a nuclear *TAS* gene which is transcribed into a mRNA and cleaved by a miRNA. The cleaved product is converted into dsRNA by the DEFECTIVE IN RNA-DIRECTED DNA METHYLATION/SUPPRESSOR OF GENE SILENCING (RDR6/SGS3) processing complex, and leading to specific siRNAs called ta-siRNAs. These mature 21-nt long siRNAs, similar to miRNAs, are able to initiate the cleavage of homologous cellular

nucleic acids, epigenetic modification and heterochromatin regulation.

such responses as well others.

mRNAs (targets) [12-13].

polymerase IV, respectively) [14-16].

**3. Non-coding small RNAs** 

### **2. An overview of beneficial interactions**

Plant-microbe interactions are not always disadvantageous to plants. During millions of years of co-evolution, plants established symbiotic interactions with bacteria and fungi. The best-studied models illustrating this kind of interaction are the rhizobial and mycorrhizal symbiosis, which involve a particular group of bacteria and fungi, respectively. The establishment of symbiosis requires a concerted molecular dialogue involving the correct recognition and the activation/repression of specific signaling pathways [6]. In the rhizobial symbiosis, *Rhizobia* form an intimate relationship with leguminous plants. Plants provide carbon and energy to the bacteria, that in exchange fix atmospheric nitrogen of interest for plants [7]. Compatible *Rhizobium* species perceive plant-secreted flavonoids and induce the expression of bacterial *nod* genes that are essential for the development of nodules in plant hosts. The Nod factors are recognized by specific plant receptors carrying an extracellular LysM domain and an intracellular kinase domain. Upon perception several cytoplasmic events occur at the root epidermal cells, including membrane depolarization, calcium spiking and activation of a calmodulin-dependent kinase signaling [7]. These processes create a favorable cellular environment leading to the establishment of an infection thread branch through which *Rhizobia* penetrate into the host. Once in the cytoplasm, bacteria group and form bacteroids where nitrogen fixation occurs. In contrast to this highly specific interaction between legumes and *Rhizobia*, mycorrhizal fungi establish symbiosis with almost all terrestrial plant species. In this case, fungi provide nutrients from the soil to the plant, particularly P, and in exchange plants feed the fungus with their photosynthetic products [8]. The nutrient transfer occurs in the arbuscules, which are specialized structures formed in cortical root cells. Arbuscular mycorrhizae (AM) depend on the activation of a symbiosis signaling (Sym) pathway, which shares some elements with rhizobial symbiosis [6]. In the case of fungal AM, the perception relies on the recognition of diffusible Myc factors, leading to the reprogramming of the basic metabolism of plant cells and hyphens.

However, these types of beneficial interactions are not always successful. Plants indeed tolerate the invasion of these microorganisms only under nitrogen or nutrient-deficient conditions. In consequence, a sophisticated perception mechanism should exist in plants in order to simultaneously estimate nutrient deficiency and distinguish between beneficial microbes and pathogens. To achieve symbiosis, a fine regulation of the plant immune responses is therefore required for accepting or not candidate microorganisms [6]. In the last years we have learned a lot about how non coding RNAs are a crucial players in regulating such responses as well others.

## **3. Non-coding small RNAs**

170 Plant Science

largest group of R proteins includes a Nucleotide Binding Site (NBS) and a LRR domain. This group can be subdivided in two subclasses based on the presence or not of a Toll-Interleukin Related (TIR) domain in the N terminus. Upon the perception of pathogen's molecules by PRRs or R proteins, a signaling cascade including MAP kinases is activated, leading to a reprogramming in host's gene expression along with the activation of genes with antimicrobial function (PR, pathogenesis related) [2, 5]. In the last years a great effort in research has focused on understanding how gene expression is modified in response to pathogens, revealing some crucial factors for the interaction such as transcription factors,

Plant-microbe interactions are not always disadvantageous to plants. During millions of years of co-evolution, plants established symbiotic interactions with bacteria and fungi. The best-studied models illustrating this kind of interaction are the rhizobial and mycorrhizal symbiosis, which involve a particular group of bacteria and fungi, respectively. The establishment of symbiosis requires a concerted molecular dialogue involving the correct recognition and the activation/repression of specific signaling pathways [6]. In the rhizobial symbiosis, *Rhizobia* form an intimate relationship with leguminous plants. Plants provide carbon and energy to the bacteria, that in exchange fix atmospheric nitrogen of interest for plants [7]. Compatible *Rhizobium* species perceive plant-secreted flavonoids and induce the expression of bacterial *nod* genes that are essential for the development of nodules in plant hosts. The Nod factors are recognized by specific plant receptors carrying an extracellular LysM domain and an intracellular kinase domain. Upon perception several cytoplasmic events occur at the root epidermal cells, including membrane depolarization, calcium spiking and activation of a calmodulin-dependent kinase signaling [7]. These processes create a favorable cellular environment leading to the establishment of an infection thread branch through which *Rhizobia* penetrate into the host. Once in the cytoplasm, bacteria group and form bacteroids where nitrogen fixation occurs. In contrast to this highly specific interaction between legumes and *Rhizobia*, mycorrhizal fungi establish symbiosis with almost all terrestrial plant species. In this case, fungi provide nutrients from the soil to the plant, particularly P, and in exchange plants feed the fungus with their photosynthetic products [8]. The nutrient transfer occurs in the arbuscules, which are specialized structures formed in cortical root cells. Arbuscular mycorrhizae (AM) depend on the activation of a symbiosis signaling (Sym) pathway, which shares some elements with rhizobial symbiosis [6]. In the case of fungal AM, the perception relies on the recognition of diffusible Myc factors, leading to the

DNA regulatory elements and non-coding small RNAs.

reprogramming of the basic metabolism of plant cells and hyphens.

However, these types of beneficial interactions are not always successful. Plants indeed tolerate the invasion of these microorganisms only under nitrogen or nutrient-deficient conditions. In consequence, a sophisticated perception mechanism should exist in plants in order to simultaneously estimate nutrient deficiency and distinguish between beneficial microbes and pathogens. To achieve symbiosis, a fine regulation of the plant immune responses is therefore required for accepting or not candidate microorganisms [6]. In the last

**2. An overview of beneficial interactions** 

In eukaryotes, the endogenous regulation of gene expression is mostly dependent on the control of the RNA polymerase II by accessory proteins including activators, repressors and the mediator complex. Then, small non-coding RNAs (sncRNA) were discovered and found to be new key elements of gene expression regulation [9, 10]. sncRNAs are short molecules of typically 18 to 30 nt, involved in gene expression control, defense against other parasitic nucleic acids, epigenetic modification and heterochromatin regulation.

The best-studied sncRNAs are microRNAs (miRNAs) and small interference RNAs (siRNAs). miRNAs derive from nuclear genes. A gene coding for a miRNA (*MIRNA*) is first transcribed by the RNA polymerase II to a primary miRNA (pri-miRNA), the size of which ranges from 100 nt to several kilobases (kb). A Dicer-like (DCL) protein DCL1 in Arabidopsis along with HYPONASTIC LEAVES1 (HYL1), process the pri-miRNA into a 70 to 400 nt long precursor miRNA (pre-miRNA). This pre-miRNA forms a characteristic hairpin-like structure. A subsequent processing step involving DCL slices the pre-miRNA to form a miRNA:miRNA\* duplex (21-22 nt). The duplex is then methylated by HEN1 and exported from the nucleus to the cytoplasm where it will join an AGO protein to form the silencing complex (RISC). Only the mature miRNA strand which is usually the one with less stable 5'-end pairing, is retained in the complex, while the passenger (miRNA\*) strand is degraded. The miRNA\* degradation process remain unknown, although some family of exoribonucleases encoded by the *SMALL RNA DEGRADING NUCLEASE* (*SDN*) genes degrades mature miRNAs which could also be involved in the miRNA\* degradation [11]. The miRNA retained in the RISC complex will then guide the silencing of complementary mRNAs (targets) [12-13].

In contrast, siRNAs originate from transgenes, viruses, transposons or other RNAs that form perfectly complementary double-stranded RNA precursors (dsRNAs). In particular, virusderived siRNAs also known as virus induced RNAs (vsiRNAs), have been extensively studied in plants. From a siRNA precursor, multiple siRNAs are generated and the silencing signal can be further amplified upon the generation of secondary siRNAs subsequently processed by RNA dependent RNA polymerases (RDRs), SILENCING DEFECTIVE3 (SDE3), NRPD1a and NRPD1b (largest subunits of Pol IVa and Pol IVb isoforms of RNA polymerase IV, respectively) [14-16].

Recently, various new types of ncRNAs have been described. Among them are the transacting siRNAs (ta-siRNAs) (21-22nt) which combine both the siRNA and miRNA pathways since they originate from a nuclear *TAS* gene which is transcribed into a mRNA and cleaved by a miRNA. The cleaved product is converted into dsRNA by the DEFECTIVE IN RNA-DIRECTED DNA METHYLATION/SUPPRESSOR OF GENE SILENCING (RDR6/SGS3) processing complex, and leading to specific siRNAs called ta-siRNAs. These mature 21-nt long siRNAs, similar to miRNAs, are able to initiate the cleavage of homologous cellular transcripts, thus acting in trans. Additionally the siRNA signal from ta-siRNAs can also be amplified upon the generation of secondary siRNAs [14, 17-19]. Also reported are natural antisense transcripts-derived siRNAs (nat-siRNAs) (21-24 nt) which are cis-acting siRNAs derived from naturally occurring overlapping regions of sense and antisense transcripts [20]. The long siRNAs (lsiRNAs) (30-40 nt) are DCL1 and AGO7 dependent in their biogenesis and act by decapping or by 5'-3' degradation of target mRNAs [21]. Whilst other types of sncRNA exist, we will focus on the most common ones and report on their role(s) in plant immunity. A comparison of the types of sncRNAs discussed in this chapter can be seen in table 1.

Small Non-Coding RNAs in Plant Immunity 173

pathogen effector proteins, this scenario is reminiscent of effector-triggered susceptibility or ETS. SS proteins were previously considered as pathogenicity factors with an important function in the development of symptoms on plant hosts during infection [26]. A single SS can exert suppressor activity at different steps of the silencing pathways. One of the beststudied and more versatile SS is HC-Pro which is able to block silencing by either interfering with DCL proteins [27] and/or sequestering 21-nt siRNA duplexes [28]. The TCV P38 coat protein [29] and CMV 2b protein [30] affect the processing of dsRNA through the inactivation of DCL proteins. The P21 and P19 proteins respectively produced by the Beet yellows virus (BYV; Closterovirus, Closteroviridae) and the Tomato bushy stunt virus (TBSV, Tombusvirus, Tombusviridae), exert their function by interacting with miRNA duplexes and hairpin derived siRNAs [28, 31, 32]. Additionally, these SS interfere with the small RNAs stability by blocking the activity of the methyltransferase protein HEN1 [33, 34]. The Beet western yellows virus (BWYV; Polerovirus, Luteoviridae) P0 protein interacts via an F-box domain with AGO1 which results in its degradation, illustrating again how important components of the RNA silencing machinery are targeted

To overcome virus-deployed strategies suppressing silencing, plants evolved R proteins recognizing specific viral proteins to trigger an immune response, which can be considered as a sort of effector-triggered immunity. This R-protein dependent ETI depends on the recognition of so called Avr proteins, which can virtually be encoded by any viral codinggene. Examples of viral Avr proteins include the coat, helicase, replicase and movement proteins [36]. More than 15 anti-viral R proteins and belonging to the large class of NBS-LRRs have been characterized, including R proteins N [37], Y-1 [38] and RT4-4 [39] respectively isolated from *Nicotiana tabacum*, *Solanum tuberosum* and *Phaseolus vulgaris* and confering resistance to TMV, PVY and CMV. On the other hand, the Rx [40], HRT [41] and RCY1 [42] proteins respectively isolated from *Solanum andigena* and Arabidopsis, belong to the CC-NBS-LRR sub-class. The immunity triggered by these proteins is considered as monogenic and dominant, and manifested by an hypersensitive response (HR) [43]. Interestingly in most cases resistance of plants against virus segregates as a recessive trait, and is expressed as a cellular immunity. Remarkably, all recessive resistance genes isolated so far encode translation initiation factors [36]. As mentioned before, once the recognition of the virus is established, a re-programming in host gene expression takes place in plant host

Recent studies suggest that sncRNAs are involved in global gene expression changes during plant-virus interaction. It has been proposed that the expression of plant miRNAs targeting plant transcripts is altered in response to virus recognition with the aim of affecting viral replication and spreading. Indeed various plant miRNAs are known to be up- or downregulated following viral infection [48-52]. For example miR1885 is induced in response to infection of *Brassica rapa* by Turnip mosaic virus and is known to target a TIR-NBS-LRR (TNL) disease-resistance gene [53]. miR164 is also induced upon viral infection and its

in order to affect silencing [35].

cells [44-47]

**5. sncRNAs and viruses: new frontiers of defense** 


**Table 1.** Comparison of important features of common types of scnRNAs found in plants.

## **4. The "zigzag model" in plant-virus interactions**

The seminal work achieved on plant-virus interactions studies led to the discovery of posttranscriptional gene silencing (PTGS) as a genuine plant defense mechanism against virus. Most of the plant viruses are positive single stranded RNAs (ssRNAs). To colonize and multiply into new plant cells, virus have to replicate several thousands of times. During this process of replication and infection, RNA viruses produce double-stranded RNAs (dsRNAs). A DCL protein, usually DCL4 in Arabidopsis*,* recognizes these dsRNAs and cleaves them producing vsiRNAs. The vsiRNAs are next incorporated into the RNA-Induced Silencing Complex (RISC) where only one of the two RNA strands is retained. This RNA strand is complementary to the viral RNA and exploited to target the RNA viral molecule and degrade it. Some vsiRNAs serve as template and substrate of an RNAdependent RNA polymerase (RdRP), thereby amplifying the signal and producing more vsiRNAs [16, 22, 23]. These vsiRNAs move through the plasmodesmata of the cell-cell assuring a systemic anti-viral defense response [24, 25]. As a general plant defense mechanism against all viruses, this first branch of resistance can be considered as analogous to MAMP-triggered immunity. In this case, dsRNAs are considered as MAMPs and DCLs that recognize the dsRNAs are viewed as a sort of PRR.

In line with the "zigzag model", virus evolved strategies to overcome this first layer of immunity. As a matter of fact, viruses carry silencing suppressors (SS) are able to act at different levels of the silencing pathway [26]. Considering these suppressors as bona fide pathogen effector proteins, this scenario is reminiscent of effector-triggered susceptibility or ETS. SS proteins were previously considered as pathogenicity factors with an important function in the development of symptoms on plant hosts during infection [26]. A single SS can exert suppressor activity at different steps of the silencing pathways. One of the beststudied and more versatile SS is HC-Pro which is able to block silencing by either interfering with DCL proteins [27] and/or sequestering 21-nt siRNA duplexes [28]. The TCV P38 coat protein [29] and CMV 2b protein [30] affect the processing of dsRNA through the inactivation of DCL proteins. The P21 and P19 proteins respectively produced by the Beet yellows virus (BYV; Closterovirus, Closteroviridae) and the Tomato bushy stunt virus (TBSV, Tombusvirus, Tombusviridae), exert their function by interacting with miRNA duplexes and hairpin derived siRNAs [28, 31, 32]. Additionally, these SS interfere with the small RNAs stability by blocking the activity of the methyltransferase protein HEN1 [33, 34]. The Beet western yellows virus (BWYV; Polerovirus, Luteoviridae) P0 protein interacts via an F-box domain with AGO1 which results in its degradation, illustrating again how important components of the RNA silencing machinery are targeted in order to affect silencing [35].

172 Plant Science

seen in table 1.

Derived from Invasive nucleic

Processed by DCL, RDR, SDE,

acids (virus, transgenes)

**4. The "zigzag model" in plant-virus interactions** 

that recognize the dsRNAs are viewed as a sort of PRR.

NRPD

transcripts, thus acting in trans. Additionally the siRNA signal from ta-siRNAs can also be amplified upon the generation of secondary siRNAs [14, 17-19]. Also reported are natural antisense transcripts-derived siRNAs (nat-siRNAs) (21-24 nt) which are cis-acting siRNAs derived from naturally occurring overlapping regions of sense and antisense transcripts [20]. The long siRNAs (lsiRNAs) (30-40 nt) are DCL1 and AGO7 dependent in their biogenesis and act by decapping or by 5'-3' degradation of target mRNAs [21]. Whilst other types of sncRNA exist, we will focus on the most common ones and report on their role(s) in plant immunity. A comparison of the types of sncRNAs discussed in this chapter can be

> non-coding regions

DCL1, HYL1,

Transcribed by Depends of origin RNA pol II RNA pol II RNA pol II

HEN1

The seminal work achieved on plant-virus interactions studies led to the discovery of posttranscriptional gene silencing (PTGS) as a genuine plant defense mechanism against virus. Most of the plant viruses are positive single stranded RNAs (ssRNAs). To colonize and multiply into new plant cells, virus have to replicate several thousands of times. During this process of replication and infection, RNA viruses produce double-stranded RNAs (dsRNAs). A DCL protein, usually DCL4 in Arabidopsis*,* recognizes these dsRNAs and cleaves them producing vsiRNAs. The vsiRNAs are next incorporated into the RNA-Induced Silencing Complex (RISC) where only one of the two RNA strands is retained. This RNA strand is complementary to the viral RNA and exploited to target the RNA viral molecule and degrade it. Some vsiRNAs serve as template and substrate of an RNAdependent RNA polymerase (RdRP), thereby amplifying the signal and producing more vsiRNAs [16, 22, 23]. These vsiRNAs move through the plasmodesmata of the cell-cell assuring a systemic anti-viral defense response [24, 25]. As a general plant defense mechanism against all viruses, this first branch of resistance can be considered as analogous to MAMP-triggered immunity. In this case, dsRNAs are considered as MAMPs and DCLs

In line with the "zigzag model", virus evolved strategies to overcome this first layer of immunity. As a matter of fact, viruses carry silencing suppressors (SS) are able to act at different levels of the silencing pathway [26]. Considering these suppressors as bona fide

Targets transcripts in cis trans trans cis Binds to AGO1, AGO2 AGO1 AGO1,AGO7 AGO1?

**Table 1.** Comparison of important features of common types of scnRNAs found in plants.

siRNA miRNA ta-siRNA nat-siRNAs

non-coding regions

RDR6/SGS3, DCL, miRNAs antisense genes

DCL1, HYL1

To overcome virus-deployed strategies suppressing silencing, plants evolved R proteins recognizing specific viral proteins to trigger an immune response, which can be considered as a sort of effector-triggered immunity. This R-protein dependent ETI depends on the recognition of so called Avr proteins, which can virtually be encoded by any viral codinggene. Examples of viral Avr proteins include the coat, helicase, replicase and movement proteins [36]. More than 15 anti-viral R proteins and belonging to the large class of NBS-LRRs have been characterized, including R proteins N [37], Y-1 [38] and RT4-4 [39] respectively isolated from *Nicotiana tabacum*, *Solanum tuberosum* and *Phaseolus vulgaris* and confering resistance to TMV, PVY and CMV. On the other hand, the Rx [40], HRT [41] and RCY1 [42] proteins respectively isolated from *Solanum andigena* and Arabidopsis, belong to the CC-NBS-LRR sub-class. The immunity triggered by these proteins is considered as monogenic and dominant, and manifested by an hypersensitive response (HR) [43]. Interestingly in most cases resistance of plants against virus segregates as a recessive trait, and is expressed as a cellular immunity. Remarkably, all recessive resistance genes isolated so far encode translation initiation factors [36]. As mentioned before, once the recognition of the virus is established, a re-programming in host gene expression takes place in plant host cells [44-47]

#### **5. sncRNAs and viruses: new frontiers of defense**

Recent studies suggest that sncRNAs are involved in global gene expression changes during plant-virus interaction. It has been proposed that the expression of plant miRNAs targeting plant transcripts is altered in response to virus recognition with the aim of affecting viral replication and spreading. Indeed various plant miRNAs are known to be up- or downregulated following viral infection [48-52]. For example miR1885 is induced in response to infection of *Brassica rapa* by Turnip mosaic virus and is known to target a TIR-NBS-LRR (TNL) disease-resistance gene [53]. miR164 is also induced upon viral infection and its

induction is due to hormone-dependent specific transcriptional activation [54]. However, the effect of this differential regulation in the outcome of the interaction is not well established, as it may also result from the silencing suppression activity of the virus. This is also the case of miR168, which is induced upon infection by various viruses in different plants [49, 55]. It was shown recently that miR168 accumulation upon infection with Cymbidum ringspot virus (CymRSV) is due to the action of the p19 SS [56]. The specific targeting of miR168 by p19 may be crucial for viral infection given that this miRNA is involved in a regulatory loop with AGO1, which forms the RISC complex and in thus involved in various silencing processes [56].

Small Non-Coding RNAs in Plant Immunity 175

**Figure 1.** General model of sncRNAs role in plant-pathogen interaction. *1*.Upon recognition of PAMPs from virus and bacteria the *2*. transcription of loci coding for sncRNAs is regulated so the biogenesis pathways of either *4.* miRNAs *5.* nat-siRNAs *6.* ta-siRNAs is activated with the aims of *7.* targeting genes that when silenced would trigger defense responses (like Auxin response factors). Transcription of sncRNAs could be stopped by the plant in response to pathogens so that positive regulators of immunity (like NBS-LRR proteins) can escape miRNA regulation (not shown). *8.* Additionally, in the case of virus, vsiRNAs can be produced during viral replication and they would target viral RNAs thus producing defense. *9.* Virus and bacteria can counterattack by using effectors and silencing suppressors to disrupt silencing and in response *10.* plants could recognize these effectors via resistance proteins. Fx= bacterial effector, SS= silencing supresor, dsRNA= double-stranded RNA, PRRs= pathogen recognition receptors, R proteins = resistance protein, RISC= RNA-induced silencing complex

The best-studied miRNA induced upon bacterial infection is miR393. By comparing the gene expression profile of wild type and transgenic plants expressing several viral SS, it was elegantly demonstrated that upon treatment with flagellin (flg22), some transcripts were more abundant in transgenic plants. Among them was found a transcript coding for the Fbox auxin receptor TIR1. By RACE (rapid amplification of cDNA ends) the authors demonstrated that this particular mRNA is targeted and cleaved by miR393. The perception of flagellin by plants induced the expression of miR393 which correlates with a clear reduction of the TIR1 protein content. This led in turn to the stabilization of Aux/IAA proteins which repress auxin signaling by heterodimerization with Auxin Response Factors

It has also been suggested that miRNAs directly target viral RNAs, as it occurs in animals [57, 58]. Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59-65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59, 66]. Furthermore transgenic plants expressing dimeric or polycistronic amiRNAs directed against different viruses result in a wider spectrum of viral resistance [60, 61, 63].

The hypothesis of plant microRNAs naturally evolving to target viral genomes has long being discussed, taking in account the potential disadvantages of the miRNA pathway over the siRNA one. The miRNA pathway is not an adaptive response since the evolution of viral genomes would be fast enough to surpass the evolution of miRNAs rendering them ineffective in a very short term. However some miRNA families may be adapted to target viral genomes, as suggested by bioinformatic analysis [67, 68].

Viruses also encode miRNAs (or similar ncRNAs) directed against the plant genomes (or even their own genomes), that will use the host miRNA machinery to be processed and execute their silencing effect. This mechanism has been described only in animal infecting viruses [69]. So far, the only mechanisms found resembling viral miRNAs in plant viruses refer to sRNAs encoded by the Cauliflower mosaic virus (CaMV) that are partially complementary to regions of the Arabidopsis genome [70] and viral sRNAs that bind the RNAi machinery to divert the silencing machinery from viral promoter and coding regions [71].

A general model of the way sncRNAs mediate the interaction between plants and viruses (as well as with bacteria discussed below) can be seen in Figure 1.

## **6. The need for auxin: responses to bacteria, fungi and symbiotic microbes**

Auxin is a relatively well-known plant hormone mainly implicated in growth which acts, under particular conditions, as a repressor of salicylic acid (SA). SA is a hormone involved in the activation of plant defenses in response to biotrophic pathogens [72, 73]. It is therefore not surprising that plants, in response to microbes, have evolved sophisticated mechanism for fine-tuning of SA-mediated responses.

involved in various silencing processes [56].

resistance [60, 61, 63].

**microbes** 

induction is due to hormone-dependent specific transcriptional activation [54]. However, the effect of this differential regulation in the outcome of the interaction is not well established, as it may also result from the silencing suppression activity of the virus. This is also the case of miR168, which is induced upon infection by various viruses in different plants [49, 55]. It was shown recently that miR168 accumulation upon infection with Cymbidum ringspot virus (CymRSV) is due to the action of the p19 SS [56]. The specific targeting of miR168 by p19 may be crucial for viral infection given that this miRNA is involved in a regulatory loop with AGO1, which forms the RISC complex and in thus

It has also been suggested that miRNAs directly target viral RNAs, as it occurs in animals [57, 58]. Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59-65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59, 66]. Furthermore transgenic plants expressing dimeric or polycistronic amiRNAs directed against different viruses result in a wider spectrum of viral

The hypothesis of plant microRNAs naturally evolving to target viral genomes has long being discussed, taking in account the potential disadvantages of the miRNA pathway over the siRNA one. The miRNA pathway is not an adaptive response since the evolution of viral genomes would be fast enough to surpass the evolution of miRNAs rendering them ineffective in a very short term. However some miRNA families may be adapted to target

Viruses also encode miRNAs (or similar ncRNAs) directed against the plant genomes (or even their own genomes), that will use the host miRNA machinery to be processed and execute their silencing effect. This mechanism has been described only in animal infecting viruses [69]. So far, the only mechanisms found resembling viral miRNAs in plant viruses refer to sRNAs encoded by the Cauliflower mosaic virus (CaMV) that are partially complementary to regions of the Arabidopsis genome [70] and viral sRNAs that bind the RNAi machinery to

A general model of the way sncRNAs mediate the interaction between plants and viruses

Auxin is a relatively well-known plant hormone mainly implicated in growth which acts, under particular conditions, as a repressor of salicylic acid (SA). SA is a hormone involved in the activation of plant defenses in response to biotrophic pathogens [72, 73]. It is therefore not surprising that plants, in response to microbes, have evolved sophisticated mechanism

viral genomes, as suggested by bioinformatic analysis [67, 68].

divert the silencing machinery from viral promoter and coding regions [71].

**6. The need for auxin: responses to bacteria, fungi and symbiotic** 

(as well as with bacteria discussed below) can be seen in Figure 1.

for fine-tuning of SA-mediated responses.

**Figure 1.** General model of sncRNAs role in plant-pathogen interaction. *1*.Upon recognition of PAMPs from virus and bacteria the *2*. transcription of loci coding for sncRNAs is regulated so the biogenesis pathways of either *4.* miRNAs *5.* nat-siRNAs *6.* ta-siRNAs is activated with the aims of *7.* targeting genes that when silenced would trigger defense responses (like Auxin response factors). Transcription of sncRNAs could be stopped by the plant in response to pathogens so that positive regulators of immunity (like NBS-LRR proteins) can escape miRNA regulation (not shown). *8.* Additionally, in the case of virus, vsiRNAs can be produced during viral replication and they would target viral RNAs thus producing defense. *9.* Virus and bacteria can counterattack by using effectors and silencing suppressors to disrupt silencing and in response *10.* plants could recognize these effectors via resistance proteins. Fx= bacterial effector, SS= silencing supresor, dsRNA= double-stranded RNA, PRRs= pathogen recognition receptors, R proteins = resistance protein, RISC= RNA-induced silencing complex

The best-studied miRNA induced upon bacterial infection is miR393. By comparing the gene expression profile of wild type and transgenic plants expressing several viral SS, it was elegantly demonstrated that upon treatment with flagellin (flg22), some transcripts were more abundant in transgenic plants. Among them was found a transcript coding for the Fbox auxin receptor TIR1. By RACE (rapid amplification of cDNA ends) the authors demonstrated that this particular mRNA is targeted and cleaved by miR393. The perception of flagellin by plants induced the expression of miR393 which correlates with a clear reduction of the TIR1 protein content. This led in turn to the stabilization of Aux/IAA proteins which repress auxin signaling by heterodimerization with Auxin Response Factors

(ARF). Flagellin perception leads to a repression of auxin signaling and consequently restricted the growth *of Pseudomonas syringae* pv. tomato (*Pst*). This study provided for the first time a link between auxin response, miRNAs and MTI. In summary plants repress the auxin signaling pathway in response to bacterial hit, favoring the defenses activated by SA, compromising vegetative growth [74].

Small Non-Coding RNAs in Plant Immunity 177

relationship between miRNAs and auxin at a more indirect level [84]. Among 33 pathogenresponsive miRNAs detected during the interaction of *Populus beijingensis* with the fungus *Dothiorella gregaria*, the induction of miR393 was observed with a peak of expression at 7 days after inoculation but their levels were reduced at 14 and 21 days [85]. On the other hand, the same study showed the repression of miR160 as was previously reported in pine infected with the fungus *Cronartium quercumm* f. sp *fusiforme* [86]. In wheat interaction with *Erysiphe graminis f. sp. tritici*, the expression pattern of miR393 was less expressed in the susceptible cultivar Jingdong8 as compared to the near-isogenic resistant line Jingdong8-Pm30 [87]. These results illustrate the fact that in some cases, conserved miRNAs may play similar functions in different pathosystems. However in other cases, the expression profile and in consequence the function of miRNAs can be specific even if their sequences and targets are conserved. Altogether, these results highlight how crucial is auxin balance in plant-microbe interactions.

Fahlgreen *et al.* [77] reported on miRNAs repressed upon infection with a strain of *Pst* DC3000 inactivated in *hrcC* which is a major component of the TTSS. One of the identified miRNAs is miR825, which is predicted to target transcripts encoding a Remorin, a transcription factor of the zinc-finger homeobox family and a frataxin-related protein. These targets are known to act as positive regulators of plant defense, it is therefore expected that

A first connection between miRNAs-mediated silencing and MTI emerged from the study of Arabidopsis AGO1 mutant lines, found to be compromised in MTI [78]. These plants are characterized by a reduction in seedling growth inhibition, callose deposition, expression of MTI-markers genes and the activation of MAP kinases and ROS production upon treatment with flagellin. Also, the growth of TTSS-mutant strains was increased in these plants. These results demonstrate that AGO1, and indirectly the silencing pathway, are key elements of MTI. These phenotypes are not observed in AGO7 mutant plants, indicating that only AGO1 activity is associated with MTI. Interestingly, the involvement of miRNAs in regulating plant immunity is not restricted to MTI. It may also be associated to ETI, as exemplified by cases of plant resistance responses against fungal pathogens, where several components of the silencing machinery were showed to play a role [88]. More precisely, Arabidopsis *sgs2-1*, *sgs1-1* and *sgs-3* mutants which are defective in siRNA production, are more susceptible to various strains of *Verticillum dahliae* but not to other pathogens such as *Botrytis cinerea*, *Alternaria brassicicola* and *Plectosphaerella cucumerina*. Intriguingly, while *ago7-2*, *dcl4-2*, *nrpd1a-3* and *rdr2-4* mutants are more susceptible to *V. dahlia*, *ago1-25*, *ago1-27*, *hen1-6* and *hst-1* mutants display enhanced resistance to this pathogen. Finally, *dcl-2*, *sde3-4* and *sde3-5*

Accordingly to the "zigzag model", adapted pathogens overcome MTI to infect particular host plants. Based on the study of Arabidopsis mutants affected in genes involved in the

mutants were as susceptible as the Arabidopsis Col-0 wild type line [88].

**7. MTI and silencing: beyond miR393 and auxin** 

the miRNAs controlling them are repressed.

**8. Towards ETS** 

miR393 was shown to be induced in Arabidopsis plants inoculated with *Pst* DC3000 strain mutated in *hrcC* [75]. This strain is defective for type III secretion, unable to deliver effector proteins into the host plant cell and consequently triggers MTI. Employing a small-RNA profiling analysis, Zhang *et al.* [76] investigated the differential expression of miRNAs in plants challenged with *Pst* DC3000 *hrcC* mutant, a virulent strain of the same species carrying an empty vector and avirulent *Pst* DC3000 containing the *avrRpt2* effector [76]. Curiously, miR393 was repressed at 6 hours post-infection (hpi) and induced at 14 hpi in the three treatments. However Northern-blot experiments show an induction in all treatments and at both time points. To explain this discrepancy, the authors suggest that miR393 may regulate auxin signaling at an early stage of bacterial infection [76].

The complex interplay between auxin and miRNAs goes beyond miR393. Several reports have shown that different ARFs and auxin receptors coding genes are regulated by other miRNAs such as miR160, miR166 and miR167, not only in response to phytopathogenic bacteria and fungi but also during beneficial interactions involving *Rhizobium* or AM [72, 76- 80]. MiR167 and miR160 which target ARFs genes, are induced upon infection with different *Pst* DC3000 strains [76, 77]. MiR160 was also found to be induced in response to flg22 and bound to AGO1. Transgenic plants over-expressing miR160 show enhanced callose deposition and higher resistance to DC3000 indicating a role for miR160 as positive regulator of plant pathogen response [78].

miR393 is highly conserved and was also detected in cassava plants challenged with *Xanthomonas axonopodis* pv. manihotis, which is the causal agent of Cassava Bacterial Blight (CBB) [81]. Interestingly the expression of miR160 and miR393 is reduced during the infection of Arabidopsis with *Agrobacterium tumefaciens*, thereby increasing auxin signaling [82].

During the symbiosis occurring between soybean and *Bradirhizobium japonicum*, miR160, which targets the auxin repressor ARF17, is down-regulated, suggesting an increase in free auxin during this interaction. In contrast, miR393 was found to be induced, which is in opposition to miR160 effect, as miR393 regulates the auxin receptor TIR1 and consequently inhibits auxin signaling [83]. In AM symbiosis, a strong connection between miRNAs and auxin has also been unveiled. During the interaction of *M. truncatula* plants with *Glomus intraradices*, it was reported that miR160c and miR167 are induced in mycorrhizal roots while miR160 was predominantly localized in the phloem [84]. These authors reported also miR5229a/b to be the most induced miRNA. By in situ hybridization it was demonstrated that it was exclusively expressed in arbuscule-containing cells of the root cortex, albeit with different signal intensities indicating a specific function during different stages of the arbuscule development. The predicted target of miR5229a/b is a transcript encoding for a heme peroxidase playing different roles in the regulation of ROS production, cell wall biosynthesis but also auxin and ethylene metabolism. This provides another example of the relationship between miRNAs and auxin at a more indirect level [84]. Among 33 pathogenresponsive miRNAs detected during the interaction of *Populus beijingensis* with the fungus *Dothiorella gregaria*, the induction of miR393 was observed with a peak of expression at 7 days after inoculation but their levels were reduced at 14 and 21 days [85]. On the other hand, the same study showed the repression of miR160 as was previously reported in pine infected with the fungus *Cronartium quercumm* f. sp *fusiforme* [86]. In wheat interaction with *Erysiphe graminis f. sp. tritici*, the expression pattern of miR393 was less expressed in the susceptible cultivar Jingdong8 as compared to the near-isogenic resistant line Jingdong8-Pm30 [87]. These results illustrate the fact that in some cases, conserved miRNAs may play similar functions in different pathosystems. However in other cases, the expression profile and in consequence the function of miRNAs can be specific even if their sequences and targets are conserved. Altogether, these results highlight how crucial is auxin balance in plant-microbe interactions.

## **7. MTI and silencing: beyond miR393 and auxin**

Fahlgreen *et al.* [77] reported on miRNAs repressed upon infection with a strain of *Pst* DC3000 inactivated in *hrcC* which is a major component of the TTSS. One of the identified miRNAs is miR825, which is predicted to target transcripts encoding a Remorin, a transcription factor of the zinc-finger homeobox family and a frataxin-related protein. These targets are known to act as positive regulators of plant defense, it is therefore expected that the miRNAs controlling them are repressed.

A first connection between miRNAs-mediated silencing and MTI emerged from the study of Arabidopsis AGO1 mutant lines, found to be compromised in MTI [78]. These plants are characterized by a reduction in seedling growth inhibition, callose deposition, expression of MTI-markers genes and the activation of MAP kinases and ROS production upon treatment with flagellin. Also, the growth of TTSS-mutant strains was increased in these plants. These results demonstrate that AGO1, and indirectly the silencing pathway, are key elements of MTI. These phenotypes are not observed in AGO7 mutant plants, indicating that only AGO1 activity is associated with MTI. Interestingly, the involvement of miRNAs in regulating plant immunity is not restricted to MTI. It may also be associated to ETI, as exemplified by cases of plant resistance responses against fungal pathogens, where several components of the silencing machinery were showed to play a role [88]. More precisely, Arabidopsis *sgs2-1*, *sgs1-1* and *sgs-3* mutants which are defective in siRNA production, are more susceptible to various strains of *Verticillum dahliae* but not to other pathogens such as *Botrytis cinerea*, *Alternaria brassicicola* and *Plectosphaerella cucumerina*. Intriguingly, while *ago7-2*, *dcl4-2*, *nrpd1a-3* and *rdr2-4* mutants are more susceptible to *V. dahlia*, *ago1-25*, *ago1-27*, *hen1-6* and *hst-1* mutants display enhanced resistance to this pathogen. Finally, *dcl-2*, *sde3-4* and *sde3-5* mutants were as susceptible as the Arabidopsis Col-0 wild type line [88].

#### **8. Towards ETS**

176 Plant Science

compromising vegetative growth [74].

regulator of plant pathogen response [78].

(ARF). Flagellin perception leads to a repression of auxin signaling and consequently restricted the growth *of Pseudomonas syringae* pv. tomato (*Pst*). This study provided for the first time a link between auxin response, miRNAs and MTI. In summary plants repress the auxin signaling pathway in response to bacterial hit, favoring the defenses activated by SA,

miR393 was shown to be induced in Arabidopsis plants inoculated with *Pst* DC3000 strain mutated in *hrcC* [75]. This strain is defective for type III secretion, unable to deliver effector proteins into the host plant cell and consequently triggers MTI. Employing a small-RNA profiling analysis, Zhang *et al.* [76] investigated the differential expression of miRNAs in plants challenged with *Pst* DC3000 *hrcC* mutant, a virulent strain of the same species carrying an empty vector and avirulent *Pst* DC3000 containing the *avrRpt2* effector [76]. Curiously, miR393 was repressed at 6 hours post-infection (hpi) and induced at 14 hpi in the three treatments. However Northern-blot experiments show an induction in all treatments and at both time points. To explain this discrepancy, the authors suggest that miR393 may

The complex interplay between auxin and miRNAs goes beyond miR393. Several reports have shown that different ARFs and auxin receptors coding genes are regulated by other miRNAs such as miR160, miR166 and miR167, not only in response to phytopathogenic bacteria and fungi but also during beneficial interactions involving *Rhizobium* or AM [72, 76- 80]. MiR167 and miR160 which target ARFs genes, are induced upon infection with different *Pst* DC3000 strains [76, 77]. MiR160 was also found to be induced in response to flg22 and bound to AGO1. Transgenic plants over-expressing miR160 show enhanced callose deposition and higher resistance to DC3000 indicating a role for miR160 as positive

miR393 is highly conserved and was also detected in cassava plants challenged with *Xanthomonas axonopodis* pv. manihotis, which is the causal agent of Cassava Bacterial Blight (CBB) [81]. Interestingly the expression of miR160 and miR393 is reduced during the infection

During the symbiosis occurring between soybean and *Bradirhizobium japonicum*, miR160, which targets the auxin repressor ARF17, is down-regulated, suggesting an increase in free auxin during this interaction. In contrast, miR393 was found to be induced, which is in opposition to miR160 effect, as miR393 regulates the auxin receptor TIR1 and consequently inhibits auxin signaling [83]. In AM symbiosis, a strong connection between miRNAs and auxin has also been unveiled. During the interaction of *M. truncatula* plants with *Glomus intraradices*, it was reported that miR160c and miR167 are induced in mycorrhizal roots while miR160 was predominantly localized in the phloem [84]. These authors reported also miR5229a/b to be the most induced miRNA. By in situ hybridization it was demonstrated that it was exclusively expressed in arbuscule-containing cells of the root cortex, albeit with different signal intensities indicating a specific function during different stages of the arbuscule development. The predicted target of miR5229a/b is a transcript encoding for a heme peroxidase playing different roles in the regulation of ROS production, cell wall biosynthesis but also auxin and ethylene metabolism. This provides another example of the

of Arabidopsis with *Agrobacterium tumefaciens*, thereby increasing auxin signaling [82].

regulate auxin signaling at an early stage of bacterial infection [76].

Accordingly to the "zigzag model", adapted pathogens overcome MTI to infect particular host plants. Based on the study of Arabidopsis mutants affected in genes involved in the silencing pathway like *dcl* and *hen1*, it could be demonstrated that non-pathogenic bacteria like *P. fluorescens* and *E. coli* were able to grow in these plants and not in the wild type. In addition, an increase in growth was observed upon inoculation of silencing-defective mutant lines with a TTSS-mutant *Pst* DC3000 strain and the non-host pathogen *Pseudomonas phaseolicola* [89]. These ground-breaking observations suggested a pivotal role of silencing in triggering MTI responses. In consequence, adapted pathogens should have acquired effector proteins capable of suppressing silencing-associated MTI pathways. As a matter of fact, the virulent strain *Pst* DC3000 was reported to repress the expression of miR393 which is normally induced upon flagellin recognition as well as other MTI responses, whereas a TTSS-mutant strain did not [89]. Since virulent *Pst* DC3000 wild type strain harbors an intact TTSS, it was concluded that miR393 repression results of the action of injected effectors. Upon *Agrobacterium*-mediated transient expression of particular T3 effectors into Arabidopsis leaves, the expression of miR393 primary transcripts was monitored. This assay successfully demonstrated that T3Es AvrPto and HOPT-1 block the miRNA pathway by targeting the activity of DCL1 and AGO, respectively [89]. Interestingly, it was also demonstrated that pre-inoculation of virus containing SS led to the development of diseaselike symptoms and favored multiplication of non-pathogenic and non-host bacteria inoculated subsequently. The authors suggest this as a molecular base explaining the synergistic interactions eventually observed between some viral and bacterial phytopathogens in the field [89].

Small Non-Coding RNAs in Plant Immunity 179

between the genes *SRRLK* and *AtRAP*. Interestingly, the biogenesis of AtlsiRNA-1 requires DCL1, DCL4, AGO7, HYL1, HEN1, HST1, RDR6 and Pol IV. The target of AtlsiRNA-1 is the gene *AtRAP*, which encodes for a RAP-domain protein with a role in plant resistance to pathogens. AtlsiRNA-1 does not cleave its target mRNA as most siRNAs usually do, but it guides their degradation through decapping and XRN4-mediated 5'-to-3' decay. A knockout mutation in *AtRAP* increases the resistance of Arabidopsis against virulent and avirulent *Pst* strains. In addition, overexpression of *AtRAP* leads to an increase in bacterial growth [91].

Since the expression of *R* genes is constitutive in most cases, it should be expected that plants have developed mechanisms regulating their activity and restrain the activation of plant immune responses under pathogen-free conditions. Although an elegant mechanism of regulation of NB-LRR proteins by conformational changes depending of the presence of the effector and hydrolysis of ATP has been described [92], controlling the activity of negative regulators mediated by sncRNAs emerges as an additional strategy to control plant

Some reports have indeed demonstrated a direct regulation of *R* genes by siRNAs. In a pioneering study, Yi and Richards [93] detected endogenous siRNAs at the *RPP5* locus with antisense transcription activity. In this locus were identified seven *R* genes of the TNL class interspersed with three related sequences and two other non *R* genes. The genes *RPP4* (Recognition of *Peronospora parasitica*, now referred to as *Hyaloperonospora arabidopsis*) and *SNC1* (suppressor of *npr1-1*, constitutive 1) present in this cluster confer resistance to fungal and bacterial pathogens, respectively and are coordinately regulated by transcription control. It was shown that a production of antisense transcripts generates siRNAs to regulate the mRNA level of these genes. In fact, in *dcl4* and *ago1* mutant Arabidopsis plants the expression of *SNC1* mRNA was elevated suggesting a role of siRNAs involved in its regulation [93]. A similar observation was reported in the symbiotic interaction of *M. truncatula* with *Shinorhizobium meliloti* where genome-wide analysis of small RNAs revealed

a relatively high proportion of 21-nt sRNAs corresponding to NBS-LRR genes [94].

Another example of regulation of *R* genes mediated by sncRNAs deals with the tobacco *N* gene which confers resistance to the tobacco mosaic virus (TMV) and codes for a TNL protein. Two miRNAs were shown to guide the cleavage of the *N* gene, namely nta-miR6019 and nta-miR6020 of 22 and 21 nt-long, respectively. In addition, a production of secondary siRNAs "in phase" with the miR6019 cleavage site of the *N* gene transcript was evidenced, and their biogenesis is dependent on DCL4 and RDR6 [95]. The co-expression of *N* with both of these nat-miRNAs led to reduced resistance against TMV confirming the importance of these nat-miRNA in the regulation of the *N* gene and N-dependent immune responses. The authors expanded these discoveries to tomato and potato, two species of the same Solanaceae family, finding that members of these miRNAs families are conserved across species as well as their potential for cleavage of NBS-LRR transcription products and the

Thus, AtRAP and PPRL can be considered negative regulators of plant immunity.

**10***. R* **genes, my favorite targets: miRNAs** 

immune responses.

generation of secondary siRNAs [95].

### **9. The arms race goes on: miRNA's role in ETI**

As mentioned previously, the specific recognition of effectors by R proteins triggers ETI, which involves gene expression reprogramming. In a survey aimed at determining the role of siRNAs in gene expression during R-protein-mediated responses it was found that a 22 nt nat-siRNA was induced specifically by *Pst* DC3000 containing the avirulence gene *avrRpt2* [90]. This nat-siRNA named nat-siRNAATGB2 is produced due to an overlapping region between the *At4g35860* and *At4g35850* transcripts. *At4g35860* encodes a Rab2-like small GTP-binding (*ATGB2)* while *At4g35850* encodes a PPR (pentatricopeptide repeats) proteinlike gene (*PPRL)*. The sequence of the nat-siRNAATGB2 is complementary to the 3' UTR region of the antisense gene *PPRL*. In fact it was demonstrated a correlation between the induced expression of nat-siRNAATGB2 and a repression of *PPRL* after infection with *Pst* carrying *avrRpt2* [90]. The induction of nat-siRNAATGB2 is dependent of the presence of *RPS2* and *NDR1*, two genes required for the induction of the *avrRpt2*-mediated response. The biogenesis of nat-siRNAATGB2 depends on the DCL1-HYL1 complex, which is stabilized upon HEN1-mediated methylation and amplified by RDR6 and SGS3. In concordance, plants mutated in these genes do not show a reduction of the *PPRL* expression. On the other hand, the overexpression of *PPRL* produced a delayed HR and enhanced growth of *Pst* DC3000 carrying *avrRpt2*, indicating that PPRL is a negative regulator of plant defense responses [90]. A novel class of sncRNAs induced in response to *Pst* DC3000 strain carrying *avrRpt2* was also identified [21]. In this case the sncRNAs are long siRNA (lsiRNA) of 30 to 40 nt. Among these lsiRNAs is AtlsiRNA-1 which is generated from a NAT pair between the genes *SRRLK* and *AtRAP*. Interestingly, the biogenesis of AtlsiRNA-1 requires DCL1, DCL4, AGO7, HYL1, HEN1, HST1, RDR6 and Pol IV. The target of AtlsiRNA-1 is the gene *AtRAP*, which encodes for a RAP-domain protein with a role in plant resistance to pathogens. AtlsiRNA-1 does not cleave its target mRNA as most siRNAs usually do, but it guides their degradation through decapping and XRN4-mediated 5'-to-3' decay. A knockout mutation in *AtRAP* increases the resistance of Arabidopsis against virulent and avirulent *Pst* strains. In addition, overexpression of *AtRAP* leads to an increase in bacterial growth [91]. Thus, AtRAP and PPRL can be considered negative regulators of plant immunity.

#### **10***. R* **genes, my favorite targets: miRNAs**

178 Plant Science

phytopathogens in the field [89].

**9. The arms race goes on: miRNA's role in ETI** 

silencing pathway like *dcl* and *hen1*, it could be demonstrated that non-pathogenic bacteria like *P. fluorescens* and *E. coli* were able to grow in these plants and not in the wild type. In addition, an increase in growth was observed upon inoculation of silencing-defective mutant lines with a TTSS-mutant *Pst* DC3000 strain and the non-host pathogen *Pseudomonas phaseolicola* [89]. These ground-breaking observations suggested a pivotal role of silencing in triggering MTI responses. In consequence, adapted pathogens should have acquired effector proteins capable of suppressing silencing-associated MTI pathways. As a matter of fact, the virulent strain *Pst* DC3000 was reported to repress the expression of miR393 which is normally induced upon flagellin recognition as well as other MTI responses, whereas a TTSS-mutant strain did not [89]. Since virulent *Pst* DC3000 wild type strain harbors an intact TTSS, it was concluded that miR393 repression results of the action of injected effectors. Upon *Agrobacterium*-mediated transient expression of particular T3 effectors into Arabidopsis leaves, the expression of miR393 primary transcripts was monitored. This assay successfully demonstrated that T3Es AvrPto and HOPT-1 block the miRNA pathway by targeting the activity of DCL1 and AGO, respectively [89]. Interestingly, it was also demonstrated that pre-inoculation of virus containing SS led to the development of diseaselike symptoms and favored multiplication of non-pathogenic and non-host bacteria inoculated subsequently. The authors suggest this as a molecular base explaining the synergistic interactions eventually observed between some viral and bacterial

As mentioned previously, the specific recognition of effectors by R proteins triggers ETI, which involves gene expression reprogramming. In a survey aimed at determining the role of siRNAs in gene expression during R-protein-mediated responses it was found that a 22 nt nat-siRNA was induced specifically by *Pst* DC3000 containing the avirulence gene *avrRpt2* [90]. This nat-siRNA named nat-siRNAATGB2 is produced due to an overlapping region between the *At4g35860* and *At4g35850* transcripts. *At4g35860* encodes a Rab2-like small GTP-binding (*ATGB2)* while *At4g35850* encodes a PPR (pentatricopeptide repeats) proteinlike gene (*PPRL)*. The sequence of the nat-siRNAATGB2 is complementary to the 3' UTR region of the antisense gene *PPRL*. In fact it was demonstrated a correlation between the induced expression of nat-siRNAATGB2 and a repression of *PPRL* after infection with *Pst* carrying *avrRpt2* [90]. The induction of nat-siRNAATGB2 is dependent of the presence of *RPS2* and *NDR1*, two genes required for the induction of the *avrRpt2*-mediated response. The biogenesis of nat-siRNAATGB2 depends on the DCL1-HYL1 complex, which is stabilized upon HEN1-mediated methylation and amplified by RDR6 and SGS3. In concordance, plants mutated in these genes do not show a reduction of the *PPRL* expression. On the other hand, the overexpression of *PPRL* produced a delayed HR and enhanced growth of *Pst* DC3000 carrying *avrRpt2*, indicating that PPRL is a negative regulator of plant defense responses [90]. A novel class of sncRNAs induced in response to *Pst* DC3000 strain carrying *avrRpt2* was also identified [21]. In this case the sncRNAs are long siRNA (lsiRNA) of 30 to 40 nt. Among these lsiRNAs is AtlsiRNA-1 which is generated from a NAT pair Since the expression of *R* genes is constitutive in most cases, it should be expected that plants have developed mechanisms regulating their activity and restrain the activation of plant immune responses under pathogen-free conditions. Although an elegant mechanism of regulation of NB-LRR proteins by conformational changes depending of the presence of the effector and hydrolysis of ATP has been described [92], controlling the activity of negative regulators mediated by sncRNAs emerges as an additional strategy to control plant immune responses.

Some reports have indeed demonstrated a direct regulation of *R* genes by siRNAs. In a pioneering study, Yi and Richards [93] detected endogenous siRNAs at the *RPP5* locus with antisense transcription activity. In this locus were identified seven *R* genes of the TNL class interspersed with three related sequences and two other non *R* genes. The genes *RPP4* (Recognition of *Peronospora parasitica*, now referred to as *Hyaloperonospora arabidopsis*) and *SNC1* (suppressor of *npr1-1*, constitutive 1) present in this cluster confer resistance to fungal and bacterial pathogens, respectively and are coordinately regulated by transcription control. It was shown that a production of antisense transcripts generates siRNAs to regulate the mRNA level of these genes. In fact, in *dcl4* and *ago1* mutant Arabidopsis plants the expression of *SNC1* mRNA was elevated suggesting a role of siRNAs involved in its regulation [93]. A similar observation was reported in the symbiotic interaction of *M. truncatula* with *Shinorhizobium meliloti* where genome-wide analysis of small RNAs revealed a relatively high proportion of 21-nt sRNAs corresponding to NBS-LRR genes [94].

Another example of regulation of *R* genes mediated by sncRNAs deals with the tobacco *N* gene which confers resistance to the tobacco mosaic virus (TMV) and codes for a TNL protein. Two miRNAs were shown to guide the cleavage of the *N* gene, namely nta-miR6019 and nta-miR6020 of 22 and 21 nt-long, respectively. In addition, a production of secondary siRNAs "in phase" with the miR6019 cleavage site of the *N* gene transcript was evidenced, and their biogenesis is dependent on DCL4 and RDR6 [95]. The co-expression of *N* with both of these nat-miRNAs led to reduced resistance against TMV confirming the importance of these nat-miRNA in the regulation of the *N* gene and N-dependent immune responses. The authors expanded these discoveries to tomato and potato, two species of the same Solanaceae family, finding that members of these miRNAs families are conserved across species as well as their potential for cleavage of NBS-LRR transcription products and the generation of secondary siRNAs [95].

During the infection of pine with the fusiform rust fungus *Cronartium quercuum*, a ta-siRNA (pta-22 ta-siRNA) targeting two disease resistance proteins was identified. In addition this study also reported and validated experimentally pta-miR946 and pta-miR948 and six of their targets which are predicted to encode for disease resistance-related transcripts, a transcript with similarity to *RPS2* and serine/treonine kinases [86]. Two other miRNAs (ptamiR950 and miR951) also target *R* genes [86]. In the response of poplar to the fungus *Dothiorella gregaria*, the targets of miRNAs pbe-miR482b, pbeSR3, pbe-SR23 and pbe-SR25 also include *R* genes. Other miRNAs previously identified in *Populus trichocarpa* include miR1447 which targets a related disease resistance-coding gene, while two other conserved miRNAs also targeting *R* genes (miR1447 and miRNA1448) are repressed [85]. Once again, these results highlight a complex network of several *R* genes whose regulation is coordinated by a huge collection of miRNAs belonging to different families and isoforms. Although miRNAs and their targets are not always validated experimentally, there is overall a clear consistency between the expression of disease resistance-related genes and their corresponding miRNAs.

Small Non-Coding RNAs in Plant Immunity 181

Besides disease resistance-related genes, other genes involved in defense pathways signaling are regulated by miRNAs. In the above-mentioned study of *Pinus taeda* infected with the fusiform rust, potential targets of a few isolated miRNAs include transcripts encoding a MYB transcription factor (pta-miR159), laccase-like genes, (miR397), peroxidases (miR420) and glutation S-transferase (GST). All these genes play a role in plant responses to pathogens, notably in gene regulation and control of ROS production [86]. Consistently, it has been shown that some of these targets are also regulated by miRNAs to avoid their expression during symbiosis. miR5282 and miRc\_275 were induced specifically in

mycorrhizal roots. These miRNAs both target *MtGst1* which encodes for a GST [84].

**11. Novel and specific miRNAs in beneficial interactions** 

whereas miR1512, miR1515 and miR1521 were only reported in soybean [96].

the invasion of pathogens.

undifferentiated cells [94].

In conclusion it appears obvious that a successful symbiosis requires suppression of host defenses. Altogether these reports stress that *R* genes are set under a multilayered and complex regulation network during interactions with microorganisms, meant to allow the establishment of beneficial interactions in favorable conditions and avoid in the mean time

sRNA studies for beneficial interactions have focused mainly on the study of legumes, and had benefited from the identification of sRNA loci in model legumes as *Lotus*, *Medicago*, *Glycine* and *Phaseolus* [98-100]. Some studies focused on the expression of specificallyinduced or repressed miRNAs during symbiosis at early [83] or late stages of the infection [72, 94]. For example, during the infection of soybean with *Bradyrhizobium japonicum*, miR168 and miR172 were induced during the first 3 hours but were gradually down regulated to reach basal levels at 12 hours. In contrast, the induction of miR159 and miR393 was sustained along the 12 hours, whereas miR160 and miR169 were down-regulated [83]. Interestingly, these studies allowed the identification of apparently specific miRNAs present or expressed only in plants able to form AM or in the symbiotic structures, respectively. Among the soybean miRNAs identified during interaction with *B. japonicum*, miR1507 seems to be legume-specific

miRNA expression was studied in soybean mutants nod49 (mutant for a Nod factor receptor *NFR1*) and nts382 (mutant for Nodule Autoregulation Receptor Kinase NARK) which are a non-nodulation and supernodulation mutants, respectively, as a result the expression of legume-specific miR1507, miR1511 and miR1512 was compromised in both mutants [96]. Another interesting and apparently specific symbiotic miRNA is miR5229a/g, which was identified in mycorrhizal roots of *M. truncatula* plants infected with *Glomus intraradices* [84]. MiR5229a/g which is the most strongly induced, was found by in situ hybridization to be exclusively expressed in arbuscules-containing cells in the root cortex, albeit with different signal intensities indicative of a specific function during different stages of the arbuscule development [84]. miR167 was localized in the differentiating peripheral vascular bundles and the novel miRNAs miR2586 and Mtr-s107 accumulate in the nodule meristem, leading the authors to conclude that miRNAs accumulate mainly in

R proteins play a pivotal role in triggering immune responses and should be able to recognize a broad spectrum of effector proteins (Jones and Dangl, 2006). The high repertory of plant immunity genes raises the question as to the control of their activity. Not surprisingly, several mechanisms were reported explaining the regulation of the expression and activity of this important type of genes. Because of the constitutive nature of many resistance genes expression, fitness costs translating into reduction of growth and productivity are significant. The regulation of *R* genes by miRNAs could have evolved as an alternative strategy for tight and cost effective regulation.

On the other hand, to achieve successful colonization, symbiotic microbes must be able to block plant immune responses triggered by non-self recognition. An expected strategy would be trough the control of plant immunity master regulators, such as *R* genes-encoded proteins and other immunity receptors. As a matter of fact, miR482 was reported to be induced during the establishment of symbiosis between soybean and *Bradyrhizobium japonicum*. Interestingly, bioinformatically-predicted targets of miR482 include various *R* genes of which two were validated experimentally. In addition, a considerable increase in the number of mature nodules was observed upon accumulation of miR482 conditionally expressed in roots under a *Rhizobium*-responsive promoter [96]. In *M. truncatula* challenged with *Shinorhizobium meliloti*, 14 targets predicted for 9 Mtr-miRNA candidates correspond to NBS-LRR coding genes [94]. Also, a high proportion of targets identified in a degradome library generated from *M. truncatula* plants infected with *Glomus intraradices* include *R* genes (27 genes) and transcription factors (33 genes). In particular it was established that miR1510a\*, miR1507, miR2678 and miR5213 regulate the expression of a subset of *R* genes [84]. More recently a deep sequencing analysis of 21 sRNAs libraries generated from four legumes (*M. truncatula*, soybean, peanut and common bean) led to the identification of several phased siRNAs (potentially ta-siRNAs), most of them targeting NBS-LRR encoding genes. These findings were expanded to potato based on bioinformatic analysis [97]. Although none of the phased siRNAs were validated by alternative experiments, the deep sequencing and the high number of libraries support well these data.

Besides disease resistance-related genes, other genes involved in defense pathways signaling are regulated by miRNAs. In the above-mentioned study of *Pinus taeda* infected with the fusiform rust, potential targets of a few isolated miRNAs include transcripts encoding a MYB transcription factor (pta-miR159), laccase-like genes, (miR397), peroxidases (miR420) and glutation S-transferase (GST). All these genes play a role in plant responses to pathogens, notably in gene regulation and control of ROS production [86]. Consistently, it has been shown that some of these targets are also regulated by miRNAs to avoid their expression during symbiosis. miR5282 and miRc\_275 were induced specifically in mycorrhizal roots. These miRNAs both target *MtGst1* which encodes for a GST [84].

In conclusion it appears obvious that a successful symbiosis requires suppression of host defenses. Altogether these reports stress that *R* genes are set under a multilayered and complex regulation network during interactions with microorganisms, meant to allow the establishment of beneficial interactions in favorable conditions and avoid in the mean time the invasion of pathogens.

## **11. Novel and specific miRNAs in beneficial interactions**

180 Plant Science

their corresponding miRNAs.

alternative strategy for tight and cost effective regulation.

sequencing and the high number of libraries support well these data.

During the infection of pine with the fusiform rust fungus *Cronartium quercuum*, a ta-siRNA (pta-22 ta-siRNA) targeting two disease resistance proteins was identified. In addition this study also reported and validated experimentally pta-miR946 and pta-miR948 and six of their targets which are predicted to encode for disease resistance-related transcripts, a transcript with similarity to *RPS2* and serine/treonine kinases [86]. Two other miRNAs (ptamiR950 and miR951) also target *R* genes [86]. In the response of poplar to the fungus *Dothiorella gregaria*, the targets of miRNAs pbe-miR482b, pbeSR3, pbe-SR23 and pbe-SR25 also include *R* genes. Other miRNAs previously identified in *Populus trichocarpa* include miR1447 which targets a related disease resistance-coding gene, while two other conserved miRNAs also targeting *R* genes (miR1447 and miRNA1448) are repressed [85]. Once again, these results highlight a complex network of several *R* genes whose regulation is coordinated by a huge collection of miRNAs belonging to different families and isoforms. Although miRNAs and their targets are not always validated experimentally, there is overall a clear consistency between the expression of disease resistance-related genes and

R proteins play a pivotal role in triggering immune responses and should be able to recognize a broad spectrum of effector proteins (Jones and Dangl, 2006). The high repertory of plant immunity genes raises the question as to the control of their activity. Not surprisingly, several mechanisms were reported explaining the regulation of the expression and activity of this important type of genes. Because of the constitutive nature of many resistance genes expression, fitness costs translating into reduction of growth and productivity are significant. The regulation of *R* genes by miRNAs could have evolved as an

On the other hand, to achieve successful colonization, symbiotic microbes must be able to block plant immune responses triggered by non-self recognition. An expected strategy would be trough the control of plant immunity master regulators, such as *R* genes-encoded proteins and other immunity receptors. As a matter of fact, miR482 was reported to be induced during the establishment of symbiosis between soybean and *Bradyrhizobium japonicum*. Interestingly, bioinformatically-predicted targets of miR482 include various *R* genes of which two were validated experimentally. In addition, a considerable increase in the number of mature nodules was observed upon accumulation of miR482 conditionally expressed in roots under a *Rhizobium*-responsive promoter [96]. In *M. truncatula* challenged with *Shinorhizobium meliloti*, 14 targets predicted for 9 Mtr-miRNA candidates correspond to NBS-LRR coding genes [94]. Also, a high proportion of targets identified in a degradome library generated from *M. truncatula* plants infected with *Glomus intraradices* include *R* genes (27 genes) and transcription factors (33 genes). In particular it was established that miR1510a\*, miR1507, miR2678 and miR5213 regulate the expression of a subset of *R* genes [84]. More recently a deep sequencing analysis of 21 sRNAs libraries generated from four legumes (*M. truncatula*, soybean, peanut and common bean) led to the identification of several phased siRNAs (potentially ta-siRNAs), most of them targeting NBS-LRR encoding genes. These findings were expanded to potato based on bioinformatic analysis [97]. Although none of the phased siRNAs were validated by alternative experiments, the deep sRNA studies for beneficial interactions have focused mainly on the study of legumes, and had benefited from the identification of sRNA loci in model legumes as *Lotus*, *Medicago*, *Glycine* and *Phaseolus* [98-100]. Some studies focused on the expression of specificallyinduced or repressed miRNAs during symbiosis at early [83] or late stages of the infection [72, 94]. For example, during the infection of soybean with *Bradyrhizobium japonicum*, miR168 and miR172 were induced during the first 3 hours but were gradually down regulated to reach basal levels at 12 hours. In contrast, the induction of miR159 and miR393 was sustained along the 12 hours, whereas miR160 and miR169 were down-regulated [83]. Interestingly, these studies allowed the identification of apparently specific miRNAs present or expressed only in plants able to form AM or in the symbiotic structures, respectively. Among the soybean miRNAs identified during interaction with *B. japonicum*, miR1507 seems to be legume-specific whereas miR1512, miR1515 and miR1521 were only reported in soybean [96].

miRNA expression was studied in soybean mutants nod49 (mutant for a Nod factor receptor *NFR1*) and nts382 (mutant for Nodule Autoregulation Receptor Kinase NARK) which are a non-nodulation and supernodulation mutants, respectively, as a result the expression of legume-specific miR1507, miR1511 and miR1512 was compromised in both mutants [96]. Another interesting and apparently specific symbiotic miRNA is miR5229a/g, which was identified in mycorrhizal roots of *M. truncatula* plants infected with *Glomus intraradices* [84]. MiR5229a/g which is the most strongly induced, was found by in situ hybridization to be exclusively expressed in arbuscules-containing cells in the root cortex, albeit with different signal intensities indicative of a specific function during different stages of the arbuscule development [84]. miR167 was localized in the differentiating peripheral vascular bundles and the novel miRNAs miR2586 and Mtr-s107 accumulate in the nodule meristem, leading the authors to conclude that miRNAs accumulate mainly in undifferentiated cells [94].

As stated before, rhizobacteria and AM share several elements of their symbiosis pathway [6] and miRNAs-mediated regulation is not an exception. miR169 which targets the CCAAT-binding transcription factor MtHAP2-1, was identified in the symbiotic interaction occuring between *Glycine max* and *B. japonicum* [83]. This transcription factor is highly induced during symbiosis and its degradation is mediated by miR169, causing a delayed nodule development and subsequent inability to fix N2 [101]. This miRNA was also found to be up-regulated in *Medicago* interacting with AM, accumulating in the phloem and around fungal hyphae [84]. Another crosstalk must be established between symbiosis and nutrition pathways to determinate if the colonization of microbes occurs or not. An illustration of this comes from miR167 and miR5204 which are up-regulated in *Medicago* mycorrhizal roots under low phosphate conditions, as compared to nonmycorrhizal roots [84]. However these miRNAs were also regulated by phosphate, pointing to a direct connection between nutrition and symbiosis. Previous studies demonstrated an induction of miR399 under Pi depleted conditions in mycorrhizal *M. truncatula* and tobacco plants associated with a concomitant increase in Pi content [102]

Small Non-Coding RNAs in Plant Immunity 183

**Author details** 

**13. References** 

14(4): 451-7.

116(2): 281-97.

Plant cell. 1990; 2(4): 279-89.

expression. The Plant cell. 1990; 2(4): 291-9.

in Arabidopsis. Science. 2008; 321(5895): 1490-2.

Boris Szurek

Camilo López and Álvaro L. Perez-Quintero

*pour le Développement, Montpellier Cedex 5, France* 

Arabidopsis. Cell. 2002; 108(6): 743-54.

plants. Annual review of plant biology. 2006; 57: 19-53.

evolution of the plant immune response. Cell. 2006; 124(4): 803-14.

Molecular plant-microbe interactions : MPMI. 2012; 25(2): 139-50.

nodule symbiosis. Current opinion in plant biology. 2011; 14(4): 458-67.

*Universidad Nacional de Colombia, Bogotá, Departamento de Biología, Bogota D.C.,Colombia* 

*UMR 186 IRD-UM2-Cirad, Résistance des Plantes aux Bioagresseurs (RPB), Institut de Recherche* 

[1] Jones-Rhoades MW, Bartel DP, Bartel B. MicroRNAS and their regulatory roles in

[2] Chisholm ST, Coaker G, Day B, Staskawicz BJ. Host-microbe interactions: shaping the

[3] Boller T, He SY. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science. 2009; 324(5928): 742-4. [4] Mackey D, Holt BF, 3rd, Wiig A, Dangl JL. RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in

[5] Tsuda K, Katagiri F. Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity. Current opinion in plant biology. 2010; 13(4): 459-65. [6] Zamioudis C, Pieterse CM. Modulation of host immunity by beneficial microbes.

[7] Popp C, Ott T. Regulation of signal transduction and bacterial infection during root

[8] Bonfante P, Requena N. Dating in the dark: how roots respond to fungal signals to establish arbuscular mycorrhizal symbiosis. Current opinion in plant biology. 2011;

[9] Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. The

[10] van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene

[11] Ramachandran V, Chen X. Degradation of microRNAs by a family of exoribonucleases

[12] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;

[13] Zhu JK. Reconstituting plant miRNA biogenesis. Proceedings of the National Academy

[14] Jamalkandi SA, Masoudi-Nejad A. Reconstruction of Arabidopsis thaliana fully integrated small RNA pathway. Functional & integrative genomics. 2009; 9(4): 419-32.

of Sciences of the United States of America. 2008; 105(29): 9851-2.

#### **12. Conclusions and perspectives**

Research in sncRNAs is ultimately one of the most active and promising fields in plant biology, and it is expected to grow even more in importance in the near future, however many aspects of sncRNAs functions during plant-microbe interactions still remain unclear. How are these sncRNAs regulated? Are there common regulatory and feedback regulatory circuits between the different classes of sncRNAs? Are there core sncRNAs and targets for different class of pathogens and for different plant species? What is the evolutionary history of these different families of sncRNAs and how did they shape plant evolution? How did differences in sncRNA regulation across the plant kingdom arise? How are new sncRNA-specificities generated and how variable can these molecules be within species or populations? Studies reviewed here highlight the importance of sncRNAs in gene regulation in response of plants to pathogens as diverse as viruses, bacteria and fungi. Some sncRNAs have been shown to be induced or repressed in response to these diverse pathogens during incompatible and compatible interactions indicating a dual role of these RNAs as positive and negative regulators of plant immunity. This fact demonstrates the complex network of gene expression during plant-microbe interactions and should be considered in biotechnological programs focused to enhance the crop resistance to plant diseases. The notable repression of *R* genes during symbiotic interactions stresses the importance of these molecules during plant-microbe interactions and provides a bridge between pathogenic and beneficial interactions. The role that effectors have and its interaction with the plant silencing machinery reveals also the amazing and surprising mechanism that pathogens have evolved to surpass the plant immunity mechanisms. Deepening on all this knowledge surely will open new ways to improve resistance against biotic stress in several plants including crops of economical importance.

#### **Author details**

Camilo López and Álvaro L. Perez-Quintero

*Universidad Nacional de Colombia, Bogotá, Departamento de Biología, Bogota D.C.,Colombia* 

Boris Szurek

182 Plant Science

concomitant increase in Pi content [102]

**12. Conclusions and perspectives** 

importance.

As stated before, rhizobacteria and AM share several elements of their symbiosis pathway [6] and miRNAs-mediated regulation is not an exception. miR169 which targets the CCAAT-binding transcription factor MtHAP2-1, was identified in the symbiotic interaction occuring between *Glycine max* and *B. japonicum* [83]. This transcription factor is highly induced during symbiosis and its degradation is mediated by miR169, causing a delayed nodule development and subsequent inability to fix N2 [101]. This miRNA was also found to be up-regulated in *Medicago* interacting with AM, accumulating in the phloem and around fungal hyphae [84]. Another crosstalk must be established between symbiosis and nutrition pathways to determinate if the colonization of microbes occurs or not. An illustration of this comes from miR167 and miR5204 which are up-regulated in *Medicago* mycorrhizal roots under low phosphate conditions, as compared to nonmycorrhizal roots [84]. However these miRNAs were also regulated by phosphate, pointing to a direct connection between nutrition and symbiosis. Previous studies demonstrated an induction of miR399 under Pi depleted conditions in mycorrhizal *M. truncatula* and tobacco plants associated with a

Research in sncRNAs is ultimately one of the most active and promising fields in plant biology, and it is expected to grow even more in importance in the near future, however many aspects of sncRNAs functions during plant-microbe interactions still remain unclear. How are these sncRNAs regulated? Are there common regulatory and feedback regulatory circuits between the different classes of sncRNAs? Are there core sncRNAs and targets for different class of pathogens and for different plant species? What is the evolutionary history of these different families of sncRNAs and how did they shape plant evolution? How did differences in sncRNA regulation across the plant kingdom arise? How are new sncRNA-specificities generated and how variable can these molecules be within species or populations? Studies reviewed here highlight the importance of sncRNAs in gene regulation in response of plants to pathogens as diverse as viruses, bacteria and fungi. Some sncRNAs have been shown to be induced or repressed in response to these diverse pathogens during incompatible and compatible interactions indicating a dual role of these RNAs as positive and negative regulators of plant immunity. This fact demonstrates the complex network of gene expression during plant-microbe interactions and should be considered in biotechnological programs focused to enhance the crop resistance to plant diseases. The notable repression of *R* genes during symbiotic interactions stresses the importance of these molecules during plant-microbe interactions and provides a bridge between pathogenic and beneficial interactions. The role that effectors have and its interaction with the plant silencing machinery reveals also the amazing and surprising mechanism that pathogens have evolved to surpass the plant immunity mechanisms. Deepening on all this knowledge surely will open new ways to improve resistance against biotic stress in several plants including crops of economical *UMR 186 IRD-UM2-Cirad, Résistance des Plantes aux Bioagresseurs (RPB), Institut de Recherche pour le Développement, Montpellier Cedex 5, France* 

#### **13. References**


[15] Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS. Post-transcriptional gene silencing by siRNAs and miRNAs. Current opinion in structural biology. 2005; 15(3): 331-41.

Small Non-Coding RNAs in Plant Immunity 185

[31] Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes &

[32] Omarov R, Sparks K, Smith L, Zindovic J, Scholthof HB. Biological relevance of a stable biochemical interaction between the tombusvirus-encoded P19 and short interfering

[33] Lozsa R, Csorba T, Lakatos L, Burgyan J. Inhibition of 3' modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins. Nucleic acids research. 2008; 36(12): 4099-107. [34] Yu B, Chapman EJ, Yang Z, Carrington JC, Chen X. Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis. FEBS

[35] Bortolamiol D, Pazhouhandeh M, Marrocco K, Genschik P, Ziegler-Graff V. The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

[36] Fraile A, Garcia-Arenal F. The coevolution of plants and viruses: resistance and

[37] Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B. The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor.

[38] Vidal S, Cabrera H, Andersson RA, Fredriksson A, Valkonen JP. Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y. Molecular

[39] Seo YS, Rojas MR, Lee JY, Lee SW, Jeon JS, Ronald P, Lucas WJ, Gilbertson RL. A viral resistance gene from common bean functions across plant families and is up-regulated in a non-virus-specific manner. Proceedings of the National Academy of Sciences of the

[40] Bendahmane A, Kanyuka K, Baulcombe DC. The Rx gene from potato controls separate

[41] Cooley MB, Pathirana S, Wu HJ, Kachroo P, Klessig DF. Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete

[42] Takahashi H, Miller J, Nozaki Y, Takeda M, Shah J, Hase S, Ikegami M, Ehara Y, Dinesh-Kumar SP. RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene, conferring resistance to cucumber mosaic virus requires salicylic acid, ethylene and a novel signal transduction mechanism. The Plant journal : for cell and molecular biology.

[43] Palukaitis P, Carr JP, Schoelz JE. Plant-virus interactions. Methods Mol Biol. 2008; 451:

[44] Marathe R, Guan Z, Anandalakshmi R, Zhao H, Dinesh-Kumar SP. Study of Arabidopsis thaliana resistome in response to cucumber mosaic virus infection using

whole genome microarray. Plant molecular biology. 2004; 55(4): 501-20.

virus resistance and cell death responses. The Plant cell. 1999; 11(5): 781-92.

development. 2004; 18(10): 1179-86.

letters. 2006; 580(13): 3117-20.

Cell. 1994; 78(6): 1101-15.

2002; 32(5): 655-67.

3-19.

RNAs. Journal of virology. 2006; 80(6): 3000-8.

Current biology : CB. 2007; 17(18): 1615-21.

pathogenicity. Advances in virus research. 2010; 76: 1-32.

plant-microbe interactions : MPMI. 2002; 15(7): 717-27.

United States of America. 2006; 103(32): 11856-61.

pathogens. The Plant cell. 2000; 12(5): 663-76.


[31] Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes & development. 2004; 18(10): 1179-86.

184 Plant Science

[15] Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS. Post-transcriptional gene silencing by siRNAs and miRNAs. Current opinion in structural biology. 2005; 15(3): 331-41. [16] Llave C. Virus-derived small interfering RNAs at the core of plant-virus interactions.

[17] Allen E, Xie Z, Gustafson AM, Carrington JC. microRNA-directed phasing during

[18] Yoshikawa M, Peragine A, Park MY, Poethig RS. A pathway for the biogenesis of trans-

[19] Guleria P, Mahajan M, Bhardwaj J, Yadav SK. Plant Small RNAs: Biogenesis, Mode of Action and Their Roles in Abiotic Stresses. Genomics, Proteomics & Bioinformatics.

[20] Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell. 2005;

[21] Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H. A novel class of bacteria-induced

[22] Garcia-Ruiz H, Takeda A, Chapman EJ, Sullivan CM, Fahlgren N, Brempelis KJ, Carrington JC. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus

[23] Voinnet O. Use, tolerance and avoidance of amplified RNA silencing by plants. Trends

[24] Palauqui JC, Balzergue S. Activation of systemic acquired silencing by localised

[25] Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient

[26] Voinnet O, Pinto YM, Baulcombe DC. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy

[27] Dunoyer P, Lecellier CH, Parizotto EA, Himber C, Voinnet O. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.

[28] Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu YP, Lopez-Moya JJ, Burgyan J. Small RNA binding is a common strategy to suppress RNA silencing by

[29] Thomas CL, Leh V, Lederer C, Maule AJ. Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology. 2003; 306(1): 33-41. [30] Diaz-Pendon JA, Li F, Li WX, Ding SW. Suppression of antiviral silencing by cucumber mosaic virus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viral small interfering RNAs. The Plant cell. 2007; 19(6):

small RNAs in Arabidopsis. Genes & development. 2007; 21(23): 3123-34.

acting siRNAs in Arabidopsis. Genes & development. 2005; 19(18): 2164-75.

trans-acting siRNA biogenesis in plants. Cell. 2005; 121(2): 207-21.

Trends in plant science. 2010; 15(12): 701-7.

infection. The Plant cell. 2010; 22(2): 481-96.

introduction of DNA. Current biology : CB. 1999; 9(2): 59-66.

of Sciences of the United States of America. 1999; 96(24): 14147-52.

several viral suppressors. The EMBO journal. 2006; 25(12): 2768-80.

in plant science. 2008; 13(7): 317-28.

cells. Science. 2010; 328(5980): 872-5.

The Plant cell. 2004; 16(5): 1235-50.

2053-63.

2011; 9(6): 183-99.

123(7): 1279-91.


[45] Espinoza C, Vega A, Medina C, Schlauch K, Cramer G, Arce-Johnson P. Gene expression associated with compatible viral diseases in grapevine cultivars. Functional & integrative genomics. 2007; 7(2): 95-110.

Small Non-Coding RNAs in Plant Immunity 187

[58] Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, Saib A, Voinnet O. A cellular microRNA mediates antiviral defense in human cells. Science.

[59] Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nature

[60] Qu J, Ye J, Fang R. Artificial microRNA-mediated virus resistance in plants. Journal of

[61] Duan CG, Wang CH, Fang RX, Guo HS. Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants. Journal of virology. 2008; 82(22): 11084-

[62] Lin SS, Wu HW, Elena SF, Chen KC, Niu QW, Yeh SD, Chen CC, Chua NH. Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-

[63] Fahim M, Millar AA, Wood CC, Larkin PJ. Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat. Plant

[64] Jelly NS, Schellenbaum P, Walter B, Maillot P. Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in

[65] Ai T, Zhang L, Gao Z, Zhu CX, Guo X. Highly efficient virus resistance mediated by artificial microRNAs that target the suppressor of PVX and PVY in plants. Plant Biol

[66] Zhang X, Li H, Zhang J, Zhang C, Gong P, Ziaf K, Xiao F, Ye Z. Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous

[67] Perez-Quintero AL, Neme R, Zapata A, Lopez C. Plant microRNAs and their role in defense against viruses: a bioinformatics approach. BMC plant biology. 2010; 10: 138. [68] Naqvi AR, Choudhury NR, Mukherjee SK, Haq QM. In silico analysis reveals that several tomato microRNA/microRNA\* sequences exhibit propensity to bind to tomato leaf curl virus (ToLCV) associated genomes and most of their encoded open reading frames (ORFs). Plant physiology and biochemistry : PPB / Societe francaise de

[69] Boss IW, Renne R. Viral miRNAs: tools for immune evasion. Current opinion in

[70] Moissiard G, Voinnet O. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proceedings of the National Academy of Sciences of the United States of America. 2006; 103(51): 19593-

[71] Blevins T, Rajeswaran R, Aregger M, Borah BK, Schepetilnikov M, Baerlocher L, Farinelli L, Meins F, Jr., Hohn T, Pooggin MM. Massive production of small RNAs from

mediated silencing. PLoS pathogens. 2009; 5(2): e1000312.

grapevine somatic embryos. Transgenic research. 2012.

manner. Transgenic research. 2011; 20(3): 569-81.

physiologie vegetale. 2011; 49(1): 13-7.

microbiology. 2010; 13(4): 540-5.

8.

biotechnology journal. 2012; 10(2): 150-63.

2005; 308(5721): 557-60.

biotechnology. 2006; 24(11): 1420-8.

virology. 2007; 81(12): 6690-9.

(Stuttg). 2011; 13(2): 304-16.

95.


[58] Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, Saib A, Voinnet O. A cellular microRNA mediates antiviral defense in human cells. Science. 2005; 308(5721): 557-60.

186 Plant Science

2009; 9: 15.

2009; 9: 152.

18.

[45] Espinoza C, Vega A, Medina C, Schlauch K, Cramer G, Arce-Johnson P. Gene expression associated with compatible viral diseases in grapevine cultivars. Functional

[46] Uzarowska A, Dionisio G, Sarholz B, Piepho HP, Xu M, Ingvardsen CR, Wenzel G, Lubberstedt T. Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling. BMC plant biology.

[47] Gonzalez-Ibeas D, Canizares J, Aranda MA. Microarray analysis shows that recessive resistance to Watermelon mosaic virus in melon is associated with the induction of defense response genes. Molecular plant-microbe interactions : MPMI. 2012; 25(1): 107-

[48] Bazzini AA, Hopp HE, Beachy RN, Asurmendi S. Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development. Proceedings of the National Academy of Sciences of the United

[49] Csorba T, Bovi A, Dalmay T, Burgyan J. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA-

and microRNA-mediated pathways. Journal of virology. 2007; 81(21): 11768-80. [50] Feng J, Wang K, Liu X, Chen S, Chen J. The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR. Gene. 2009; 437(1-2): 14-21. [51] Naqvi AR, Haq QM, Mukherjee SK. MicroRNA profiling of tomato leaf curl New Delhi virus (tolcndv) infected tomato leaves indicates that deregulation of mir159/319 and

mir172 might be linked with leaf curl disease. Virology journal. 2010; 7: 281.

[52] Pacheco R, Garcia-Marcos A, Barajas D, Martianez J, Tenllado F. PVX-potyvirus synergistic infections differentially alter microRNA accumulation in Nicotiana

[53] He XF, Fang YY, Feng L, Guo HS. Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIR-NBS-LRR class R gene-derived novel

[54] Bazzini AA, Almasia NI, Manacorda CA, Mongelli VC, Conti G, Maroniche GA, Rodriguez MC, Distefano AJ, Hopp HE, del Vas M, Asurmendi S. Virus infection elevates transcriptional activity of miR164a promoter in plants. BMC plant biology.

[55] Havelda Z, Varallyay E, Valoczi A, Burgyan J. Plant virus infection-induced persistent host gene downregulation in systemically infected leaves. The Plant journal : for cell

[56] Varallyay E, Valoczi A, Agyi A, Burgyan J, Havelda Z. Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation. The EMBO

[57] Llave C. MicroRNAs: more than a role in plant development? Molecular plant

& integrative genomics. 2007; 7(2): 95-110.

States of America. 2007; 104(29): 12157-62.

benthamiana. Virus research. 2012; 165(2): 231-5.

and molecular biology. 2008; 55(2): 278-88.

journal. 2010; 29(20): 3507-19.

pathology. 2004; 5(4): 361-6.

miRNA in Brassica. FEBS letters. 2008; 582(16): 2445-52.


a non-coding region of Cauliflower mosaic virus in plant defense and viral counterdefense. Nucleic acids research. 2011; 39(12): 5003-14.

Small Non-Coding RNAs in Plant Immunity 189

[86] Lu S, Sun YH, Amerson H, Chiang VL. MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development. The Plant journal : for cell and

[87] Xin M, Wang Y, Yao Y, Xie C, Peng H, Ni Z, Sun Q. Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum

[88] Ellendorff U, Fradin EF, de Jonge R, Thomma BP. RNA silencing is required for Arabidopsis defence against Verticillium wilt disease. Journal of experimental botany.

[89] Navarro L, Jay F, Nomura K, He SY, Voinnet O. Suppression of the microRNA pathway

[90] Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A, Jr., Zhu JK, Staskawicz BJ, Jin H. A pathogen-inducible endogenous siRNA in plant immunity. Proceedings of the National Academy of Sciences of the United States of America. 2006;

[91] Padmanabhan C, Zhang X, Jin H. Host small RNAs are big contributors to plant innate

[92] Takken FL, Albrecht M, Tameling WI. Resistance proteins: molecular switches of plant

[93] Yi H, Richards EJ. A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing. The Plant cell. 2007; 19(9):

[94] Lelandais-Briere C, Naya L, Sallet E, Calenge F, Frugier F, Hartmann C, Gouzy J, Crespi M. Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules. The Plant cell. 2009; 21(9):

[95] Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, Sun H, Kumar P, Baker B. MicroRNA regulation of plant innate immune receptors. Proceedings of the National

[96] Li H, Deng Y, Wu T, Subramanian S, Yu O. Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation. Plant physiology. 2010; 153(4): 1759-70. [97] Zhai J, Jeong DH, De Paoli E, Park S, Rosen BD, Li Y, Gonzalez AJ, Yan Z, Kitto SL, Grusak MA. MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes & development. 2011; 25(23):

[98] Sunkar R, Jagadeeswaran G. In silico identification of conserved microRNAs in large

[99] Szittya G, Moxon S, Santos DM, Jing R, Fevereiro MP, Moulton V, Dalmay T. Highthroughput sequencing of Medicago truncatula short RNAs identifies eight new

[100] Arenas-Huertero C, Perez B, Rabanal F, Blanco-Melo D, De la Rosa C, Estrada-Navarrete G, Sanchez F, Covarrubias AA, Reyes JL. Conserved and novel miRNAs in

Academy of Sciences of the United States of America. 2012; 109(5): 1790-5.

number of diverse plant species. BMC plant biology. 2008; 8: 37.

miRNA families. BMC genomics. 2008; 9: 593.

by bacterial effector proteins. Science. 2008; 321(5891): 964-7.

immunity. Current opinion in plant biology. 2009; 12(4): 465-72.

defence. Current opinion in plant biology. 2006; 9(4): 383-90.

molecular biology. 2007; 51(6): 1077-98.

L.). BMC plant biology. 2010; 10: 123.

2009; 60(2): 591-602.

103(47): 18002-7.

2929-39.

2780-96.

2540-53.


[86] Lu S, Sun YH, Amerson H, Chiang VL. MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development. The Plant journal : for cell and molecular biology. 2007; 51(6): 1077-98.

188 Plant Science

a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-

[72] Wang Y, Li P, Cao X, Wang X, Zhang A, Li X. Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules. Biochemical and biophysical research

[73] Grant MR, Jones JD. Hormone (dis)harmony moulds plant health and disease. Science.

[74] Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling.

[75] Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL, Carrington JC. High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PloS

[76] Zhang W, Gao S, Zhou X, Chellappan P, Chen Z, Zhang X, Fromuth N, Coutino G, Coffey M, Jin H. Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks. Plant molecular biology. 2011; 75(1-2): 93-105. [77] Fahlgren N, Jogdeo S, Kasschau KD, Sullivan CM, Chapman EJ, Laubinger S, Smith LM, Dasenko M, Givan SA, Weigel D, Carrington JC. MicroRNA gene evolution in

Arabidopsis lyrata and Arabidopsis thaliana. The Plant cell. 2010; 22(4): 1074-89. [78] Li Y, Zhang Q, Zhang J, Wu L, Qi Y, Zhou JM. Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. Plant

[79] Jay F, Renou JP, Voinnet O, Navarro L. Biotic stress-associated microRNAs: identification, detection, regulation, and functional analysis. Methods Mol Biol. 2010;

[80] Jin H. Endogenous small RNAs and antibacterial immunity in plants. FEBS letters. 2008;

[81] Perez-Quintero AL, Quintero A, Urrego O, Vanegas P, Lopez C. Bioinformatic identification of cassava miRNAs differentially expressed in response to infection by

[82] Pruss GJ, Nester EW, Vance V. Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone. Molecular

[83] Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O. Novel and nodulation-

[84] Devers EA, Branscheid A, May P, Krajinski F. Stars and symbiosis: microRNA- and microRNA\*-mediated transcript cleavage involved in arbuscular mycorrhizal

[85] Chen L, Ren Y, Zhang Y, Xu J, Zhang Z, Wang Y. Genome-wide profiling of novel and conserved Populus microRNAs involved in pathogen stress response by deep

Xanthomonas axonopodis pv. manihotis. BMC plant biology. 2012; 12: 29.

regulated microRNAs in soybean roots. BMC genomics. 2008; 9: 160.

plant-microbe interactions : MPMI. 2008; 21(12): 1528-38.

symbiosis. Plant physiology. 2011; 156(4): 1990-2010.

sequencing. Planta. 2012; 235(5): 873-83.

defense. Nucleic acids research. 2011; 39(12): 5003-14.

communications. 2009; 378(4): 799-803.

2009; 324(5928): 750-2.

one. 2007; 2(2): e219.

592: 183-202.

582(18): 2679-84.

Science. 2006; 312(5772): 436-9.

physiology. 2010; 152(4): 2222-31.


the legume Phaseolus vulgaris in response to stress. Plant molecular biology. 2009; 70(4): 385-401.

**Chapter 8** 

© 2012 Pogue and Holzberg, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Pogue and Holzberg, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Transient Virus Expression Systems for** 

**Recombinant Protein Expression in** 

Gregory P. Pogue and Steven Holzberg

http://dx.doi.org/10.5772/54187

offered by transgenic systems.

**1. Introduction** 

Additional information is available at the end of the chapter

**Dicot- and Monocotyledonous Plants** 

Plants have long been a source for traditional medicinal products. Indeed, greater than four billion people utilize plants to meet their primary health care [1-2]. There are >120 distinct drugs derived from plant sources representing >70% of the approved drugs in the past 20 years [3, 4]. The manner to exploit the scale and cost advantages of agriculture while diversifying the product offerings made available by plants has been under intense investigation since the early 1980s. Traditional transgenic approaches were initially pursued, but the challenges associated with the transformation and regeneration of viable recombinant crops delayed the appearance of initial products of medicinal promise until 1989 with the production of antibodies [5] and 1990 with the production of human serum albumin [6]. During this time, the concept of using plant virus genomes as expression vectors emerged. In early investigations, researchers recognized the natural capability of virus systems to change the translational priorities within infected cells such that virally encoded proteins were produced preferentially. This ability suggested that expression vectors could be constructed from viral nucleic acids to produce recombinant proteins throughout infected plants [7]. However, for this hypothesis to be tested, the genomes of viruses, starting with positive (+) strand RNA viruses, had to be cloned and characterized [8-10]. Soon after the first full-length "infectious" clones of a (+) strand RNA plant virus were constructed, and preceding traditional transgenic systems, the virus genome was converted into an expression vector [11]. Although limited with regards *to in planta* expression, this first vector revealed the promise of virus genomes to be efficient expression systems for plants. The advantages revealed in these early studies, continue to be present: cDNA "infectious clones" offer facile subcloning vehicles allowing rapid prototyping of genetic expression constructs, and recombinant protein expression levels that exceed that


**Chapter 8** 
