**Acknowledgements**

the DSF QS system in *Xylella* is not the same as the DSF in *Xanthomonas* spp. The *Xylella* DSF signals have been characterized as cis-2-tetradecenoic acid (XfDSF1) and 2-cis-hexadecanoic acid (XfDSF2), [117, 118]. Whilst in *Xylella*, mutation of the DSF signaling results in up regulation of pathogenicity genes [114], production of cell wall degrading enzymes and expression of type IV

As noted in the discussion above, some plant pathogenic bacteria encode LuxR homologs that are capable of 'eavesdropping' by utilizing AHL mimicking low molecular weight compounds that are produced by plants. In place of the LuxI, the LuxR homologs in plant pathogenic bacteria are in most oftenly in close proximity to the *pip* gene [119]. The *pip* harbors an inverted repeat unit similar to *luxI* and is directly involved in pathogenicity, hence its biological role merits further investigation. Over the past decade, researchers have attempted to investigate these LuxR proteins especially on deciphering their role in QS signaling. The binding motifs of these LuxR homologs is unique and distinct from the conventional LuxR homolog, they lack one or two of the several conserved regions required for AHL binding [5, 8, 119]. The AHL binding domain of these proteins are substituted by methionine and tryptophan in the conserved region allowing specificity for binding to plant derived molecules [119]. The orthologs of these LuxR proteins are also encoded on the genomes of AHL producing bacteria including *Pseudomonas syringae* [8]. Consequently, questions arise, do these LuxR homologs bind to the AHL mimicking compounds and function in a similar way in the AHL producing and non AHL producing bacteria? In addition, the AHL mimicking molecules produced by plants still need to be characterized.

A variety of bacterial species are increasingly becoming resistant to the antimicrobial agents that are currently in use [120]. Resistance to streptomycin in plant pathogenic bacteria was reported within a decade of its use in controlling plant infections and diseases [121]. Research efforts are now focusing on alternative bacterial control strategies. The discovery of the involvement of QS in the regulation of bacterial virulence has led to escalated research efforts towards discovering possible biological control measures that target QS systems. The main advantage of control measures that target QS systems, though not yet scientifically proven, is

For an effective application of QS inhibition as a biological antimicrobial measure, a better understanding of the genes influenced by QS is crucial. Latest technology including research tools such as RNA-Seq has made it possible for whole transcriptome investigations to be conducted. In addition, targeted mutation and characterization of mutants has helped in unveiling the biological significance of specific genes in bacteria, the complexity of bacterial transcriptomes and thus regulation of gene expression. Nonetheless, as additional experimental and analytical tools become available, the critical role of bacterial QS to plant pathogenesis will undoubtedly become much clearer.

pili in the mutants, the opposite happens in DSF QS mutants in *Xanthomonas* spp. [6].

**3. Progress in understanding interkingdom QS**

**4. Conclusions**

78 Advances in Plant Pathology

that they are less prone to selective pressure [122].

The authors would like to thank the National Research Foundation (NRF) of South Africa, the University of Pretoria, the Forestry and Agricultural Biotechnology Institute (FABI), the Tree Protection Cooperative Program (TPCP) and Centre of Excellence in Tree Health Biotechnology (CTHB) for supporting this research.

## **Conflict of interest**

Authors declare no conflict of interest.

## **Author details**

Siphathele Sibanda1,2, Lucy Novungayo Moleleki<sup>1</sup> , Divine Yufetar Shyntum<sup>1</sup> and Teresa Ann Coutinho1,2\*

\*Address all correspondence to: teresa.coutinho@fabi.up.ac.za

1 Department of Microbiology, Faculty of Natural and Agricultural Sciences, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa

2 Faculty of Natural and Agricultural Sciences, Centre for Microbial Ecology and Genomics (CMEG), University of Pretoria, Pretoria, Republic of South Africa

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88 Advances in Plant Pathology


**Section 3**

**Advances in Viral Plant Pathology**

**Advances in Viral Plant Pathology**

**Chapter 5**

Provisional chapter

**Leaf Curl Disease: A Significant Constraint in the**

DOI: 10.5772/intechopen.76049

Tomato (Lycopersicon esculentum Mill.) is one of the most economically important vegetable crops in the world. Among the major biotic constraints, virus-associated Tomato leaf curl disease (ToLCD) is a major limiting factor affecting its cultivation and yield. Different symptoms associated with disease are reported such as leaf curling, puckering of leaves, vein yellowing, stunting, excessive branching, from pale yellowing to deep yellowing, and small leaves. The genus Begomovirus is a circular single-stranded DNA virus which is exclusively being transmitted by whitefly (Bemisia tabaci) in a persistent circulative manner. Most of the begomovirus species are monopartite (having DNA-A molecule only), except few species, which are bipartite (having DNA-A and DNA-B as the genomic component). No absolute effective control measures of the disease could be developed so far, except resistance, management of insect vectors, and altering the dates of sowing to avoid peaks of insect vector population. This chapter reports an account of history, symptoms, transmission, genome organization, distribution, and management of Tomato leaf

Keywords: Begomovirus, leaf curl, AAP, IAP, betasatellite, DNA-A, DNA-B

Tomato (Lycopersicon esculentum Mill.) is one of the most economically important vegetable crops in the world. The total area of tomato cultivation in the world is 4.582 mha with production of 150.51 MT, and China, India, the USA, Italy, Turkey, and Egypt are major

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

Leaf Curl Disease: A Significant Constraint in the

**Production of Tomato in India**

Production of Tomato in India

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Pradeep Kumar and Manish Kumar

Pradeep Kumar and Manish Kumar

http://dx.doi.org/10.5772/intechopen.76049

Abstract

curl disease.

1. Introduction

tomato-growing countries in the world.

#### **Leaf Curl Disease: A Significant Constraint in the Production of Tomato in India** Leaf Curl Disease: A Significant Constraint in the Production of Tomato in India

DOI: 10.5772/intechopen.76049

Pradeep Kumar and Manish Kumar Pradeep Kumar and Manish Kumar

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76049

#### Abstract

Tomato (Lycopersicon esculentum Mill.) is one of the most economically important vegetable crops in the world. Among the major biotic constraints, virus-associated Tomato leaf curl disease (ToLCD) is a major limiting factor affecting its cultivation and yield. Different symptoms associated with disease are reported such as leaf curling, puckering of leaves, vein yellowing, stunting, excessive branching, from pale yellowing to deep yellowing, and small leaves. The genus Begomovirus is a circular single-stranded DNA virus which is exclusively being transmitted by whitefly (Bemisia tabaci) in a persistent circulative manner. Most of the begomovirus species are monopartite (having DNA-A molecule only), except few species, which are bipartite (having DNA-A and DNA-B as the genomic component). No absolute effective control measures of the disease could be developed so far, except resistance, management of insect vectors, and altering the dates of sowing to avoid peaks of insect vector population. This chapter reports an account of history, symptoms, transmission, genome organization, distribution, and management of Tomato leaf curl disease.

Keywords: Begomovirus, leaf curl, AAP, IAP, betasatellite, DNA-A, DNA-B

## 1. Introduction

Tomato (Lycopersicon esculentum Mill.) is one of the most economically important vegetable crops in the world. The total area of tomato cultivation in the world is 4.582 mha with production of 150.51 MT, and China, India, the USA, Italy, Turkey, and Egypt are major tomato-growing countries in the world.

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

India is ranked second in area and production after China. The tomato crop covers a total area of 0.879 mha with a production of 18.22 m.t. Andhra Pradesh, Karnataka, Orissa, Maharashtra, West Bengal, Bihar, Gujarat, Chhattisgarh, Tamil Nadu, and Jharkhand are leading states [27].

genus Begomovirus, family Geminiviridae [4, 5]. This chapter summarizes the work carried out

Leaf Curl Disease: A Significant Constraint in the Production of Tomato in India

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95

The symptoms of leaf curl disease are very complex, and the typical symptoms include leaf curling, puckering of leaves, vein yellowing, stunting, excessive branching, from pale yellowing to deep yellowing, and smalling of leaves [6]. Apart from this, it also causes the extreme distortion of leaves, stunting of plants, and premature drop of flower and fruits. Singh and Lal, in 1964, observed that in some genotypes, it causes green vein banding, twisting, and green enation on the under surface of the leaf, upward rolling of margin, and islands of golden colors scattered amidst the normal green tissue [7]. The type of symptom produced is dependent on the genotype cultivated and the developmental stage at which the infection occurs. At cellular level, structural changes have been observed like hypertrophy of nucleus and accumulation of dark granules and the aggregate of virus-like particles in the cytoplasm [8] (Figure 1).

Very rich information is available regarding the transmission of the leaf curl viruses of tomato since its discovery. The virus is whitefly transmitted and was demonstrated to occur in several hosts by Vasudeva et al. as early as in 1948. As per the observation of his group, they found

on leaf curl of tomato in India.

4. Transmission of the virus

Figure 1. Showing leaf curl symptom in tomato crop.

3. Symptomatology

## 2. Leaf curl disease

Several biotic and abiotic factors are the major constraints in production of tomato in India. Among the biotic constraints, different viral diseases cause significant impact in tomato production (Table 1). Among these viral diseases, tomato leaf curl disease (ToLCD) is devastating and causes significant yield loss under severe conditions. In India, Tomato leaf curl disease (ToLCD) was first reported by Vasudeva in 1948 from Northern India and Sam Raj in 1950 from Central India. The virus can cause infection at any stage of growth and development of plants. Muniyappa et al. [8] reported that yield in summer-planted tomato is less (6.4–52.2%) as compared to winter-planted crops (52.5–100%). Disease incidence, severity, and losses occurred due to this disease depend on the time of infection and is reported to range between 17.6% and 99.7% [1, 2]. Shashti and Singh [3] reported 92.3% loss when infection occurred at 30 days after transplanting. The yield reductions were 94.9, 90.0, 78.0, and 10.8% when plants got infected in 2, 4, 6, and 10 weeks after planting [3]. The disease is caused by different species (Table 1) having circular single-stranded DNA (ssDNA), of the


Table 1. Begomovirus species associated with tomato leaf curl disease in India.

genus Begomovirus, family Geminiviridae [4, 5]. This chapter summarizes the work carried out on leaf curl of tomato in India.

## 3. Symptomatology

India is ranked second in area and production after China. The tomato crop covers a total area of 0.879 mha with a production of 18.22 m.t. Andhra Pradesh, Karnataka, Orissa, Maharashtra, West Bengal, Bihar, Gujarat, Chhattisgarh, Tamil Nadu, and Jharkhand are leading states [27].

Several biotic and abiotic factors are the major constraints in production of tomato in India. Among the biotic constraints, different viral diseases cause significant impact in tomato production (Table 1). Among these viral diseases, tomato leaf curl disease (ToLCD) is devastating and causes significant yield loss under severe conditions. In India, Tomato leaf curl disease (ToLCD) was first reported by Vasudeva in 1948 from Northern India and Sam Raj in 1950 from Central India. The virus can cause infection at any stage of growth and development of plants. Muniyappa et al. [8] reported that yield in summer-planted tomato is less (6.4–52.2%) as compared to winter-planted crops (52.5–100%). Disease incidence, severity, and losses occurred due to this disease depend on the time of infection and is reported to range between 17.6% and 99.7% [1, 2]. Shashti and Singh [3] reported 92.3% loss when infection occurred at 30 days after transplanting. The yield reductions were 94.9, 90.0, 78.0, and 10.8% when plants got infected in 2, 4, 6, and 10 weeks after planting [3]. The disease is caused by different species (Table 1) having circular single-stranded DNA (ssDNA), of the

S. No Name of the virus species Acronym Locality Year of report

 Tomato leaf curl Kerala virus ToLKeV Kerala 2011 Tomato leaf curl Ranchi virus ToLCRnV Ranchi 2011 Tomato leaf curl Patna virus ToLCPaV Patna 2010 Tomato leaf curl Rajasthan virus ToLCRV Rajasthan 2011 Tomato leaf curl Pune virus ToLCPuV Pune 2011 Tomato leaf curl Bangalore virus ToLCBV Bangalore 2000 Tomato leaf curl Karnataka virus ToLCKV Karnataka 2002 Tomato leaf curl Joydebpur virus ToLCJoV Joydebpur 2013

9 Tomato leaf curl New Delhi virus ToLCNDV New Delhi 1993 10 Tomato leaf curl Palampur virus ToLCPalV Palampur 2008 11 Tomato leaf curl Gujarat virus ToLCGV Gujarat 2003

12 Ageratum enation virus AEV Pantnagar 2013 13 Cotton leaf curl Burewala virus CLCBV Bihar 2013 14 Tobacco curly shoot virus TbCSV Pantnagar 2013

Table 1. Begomovirus species associated with tomato leaf curl disease in India.

2. Leaf curl disease

94 Advances in Plant Pathology

Monopartite and originally reported from tomato

Bipartite and originally reported from tomato

Monopartite and originally reported from tomato

The symptoms of leaf curl disease are very complex, and the typical symptoms include leaf curling, puckering of leaves, vein yellowing, stunting, excessive branching, from pale yellowing to deep yellowing, and smalling of leaves [6]. Apart from this, it also causes the extreme distortion of leaves, stunting of plants, and premature drop of flower and fruits. Singh and Lal, in 1964, observed that in some genotypes, it causes green vein banding, twisting, and green enation on the under surface of the leaf, upward rolling of margin, and islands of golden colors scattered amidst the normal green tissue [7]. The type of symptom produced is dependent on the genotype cultivated and the developmental stage at which the infection occurs. At cellular level, structural changes have been observed like hypertrophy of nucleus and accumulation of dark granules and the aggregate of virus-like particles in the cytoplasm [8] (Figure 1).

Figure 1. Showing leaf curl symptom in tomato crop.

#### 4. Transmission of the virus

Very rich information is available regarding the transmission of the leaf curl viruses of tomato since its discovery. The virus is whitefly transmitted and was demonstrated to occur in several hosts by Vasudeva et al. as early as in 1948. As per the observation of his group, they found that in winter crop the symptoms appeared 25 days post-inoculation, whereas in summer crop, it took only 15 days. Since the virus characterization was accomplished in the last decade of the twentieth century, it could be presumed that detailed studies on the virus from Southern India may represent the data for monopartite begomovirus mainly ToLCBaV and in Northern India it may include ToLCNDV. The transmission efficiency of the virus depends on the season and the prevailing temperature of the geographical location. Butter et al. [1] reported 100% transmission of virus, the virus with 10 whiteflies/plant at the temperature ranging from 33 to 39C [1]. Muniyappa et al. [9] described that geographically a different isolate of whitefly behaves differently for acquisition access period (AAP) and inoculation access period (IAP), and he reported minimum 10-min AAP (acquisition access period) and 20-minute IAP (inoculation access period), respectively, for ToLCBaV [9]. It has also recently been reported that ToLCNDV, ToLCGuV, and ToLCKaV are transmissible through sap [10–13]. The begomoviruses causing tomato leaf curl disease have a wide host range affecting various dicotyledonous plants belonging to different families. Host range of the viruses has been determined by graft/whitefly transmission, agroinoculation/biolistic delivery of viral genome into tomato plants, or by detecting the viruses in naturally infected plants using specific primers or probes to virus species.

BC1 is mainly involved in virus movement within the host [17, 18]. Betasatellite of monopartite species carries only one ORF, which is coded for BC1 protein in complementary sense.

Leaf Curl Disease: A Significant Constraint in the Production of Tomato in India

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It has been reported earlier that tomato-infecting begomoviruses have bipartite genome in North India, while monopartite genome in South India [18]. Association of betasatellite DNA

In India, the population of tomato leaf curl viruses is so diverse, and it was shown after coat protein analysis of the 29 infected tomato samples. Based on these analysis, five clusters (with less than 88% similarity among them) were observed among the population, whereas four of them represented the tomato leaf curl viruses. Out of five, one cluster showed 89% similarity with Croton yellow vein mosaic virus [20]. As of now, so many closely related tomato-infecting

molecule with the ToLCD occurring in several places of India was reported [19].

ToLCNDV is also found associated with alphasatellites (Figure 2).

begomovirus have been cloned and sequenced from India.

Figure 2. Genome organization of begomovirus associated with leaf curl disease of tomato.

### 5. Begomovirus species and its genome organization

The begomoviruses (genus Begomovirus, family Geminiviridae) constitute the largest group of plant viruses causing devastating crop diseases in India. About 16% of gemini viruses are recorded worldwide occur in India. Currently, 322 begomovirus species have officially been accepted by International Committee on Taxonomy of Viruses (ICTV) from all over the world causing infection in different crops—out of them 82 are reported from India. Among them, around 19 species of begomovirus have been shown to cause leaf curl disease in tomato (Table 1). Although the diseases were observed during the mid-twentieth century, the etiology of the disease as a begomovirus was confirmed in the last decade of the twentieth century [14]. Subsequently, based on nucleotide sequence similarity of DNA-A (<89% earlier, and <91% now) genome, different species have been identified. Two species namely Tomato leaf curl New Delhi virus and Tomato leaf curl Palampur virus predominantly distributed in Northern India and one species namely Tomato leaf curl Bangalore virus is dominant in Southern India [9, 14, 15].

The genus Begomovirus is a circular single-stranded DNA virus which is exclusively being transmitted by whitefly (Bemisia tabaci) in a persistent circulative manner [16]. Most of the begomovirus species are monopartite (having DNA-A molecule only) except few species, which are bipartite (having DNA-A and DNA-B as genomic component). However, in monopartite species, in addition to DNA-A molecule, betasatellite DNA is also present which is almost half of DNA-A component. The DNA-A of both mono- and bipartite species coding for six open reading frames (ORF), two in sense orientation namely AV1 and AV2 and four in complementary orientation namely AC1, AC2, AC3, and AC4, mainly are involved in virus replication and transmission. On the other hand, DNA-B of bipartite species coding for two ORF one in sense orientation namely BV1 and another in complementary orientation namely BC1 is mainly involved in virus movement within the host [17, 18]. Betasatellite of monopartite species carries only one ORF, which is coded for BC1 protein in complementary sense. ToLCNDV is also found associated with alphasatellites (Figure 2).

that in winter crop the symptoms appeared 25 days post-inoculation, whereas in summer crop, it took only 15 days. Since the virus characterization was accomplished in the last decade of the twentieth century, it could be presumed that detailed studies on the virus from Southern India may represent the data for monopartite begomovirus mainly ToLCBaV and in Northern India it may include ToLCNDV. The transmission efficiency of the virus depends on the season and the prevailing temperature of the geographical location. Butter et al. [1] reported 100% transmission of virus, the virus with 10 whiteflies/plant at the temperature ranging from 33 to 39C [1]. Muniyappa et al. [9] described that geographically a different isolate of whitefly behaves differently for acquisition access period (AAP) and inoculation access period (IAP), and he reported minimum 10-min AAP (acquisition access period) and 20-minute IAP (inoculation access period), respectively, for ToLCBaV [9]. It has also recently been reported that ToLCNDV, ToLCGuV, and ToLCKaV are transmissible through sap [10–13]. The begomoviruses causing tomato leaf curl disease have a wide host range affecting various dicotyledonous plants belonging to different families. Host range of the viruses has been determined by graft/whitefly transmission, agroinoculation/biolistic delivery of viral genome into tomato plants, or by detecting the viruses in naturally infected plants using specific

The begomoviruses (genus Begomovirus, family Geminiviridae) constitute the largest group of plant viruses causing devastating crop diseases in India. About 16% of gemini viruses are recorded worldwide occur in India. Currently, 322 begomovirus species have officially been accepted by International Committee on Taxonomy of Viruses (ICTV) from all over the world causing infection in different crops—out of them 82 are reported from India. Among them, around 19 species of begomovirus have been shown to cause leaf curl disease in tomato (Table 1). Although the diseases were observed during the mid-twentieth century, the etiology of the disease as a begomovirus was confirmed in the last decade of the twentieth century [14]. Subsequently, based on nucleotide sequence similarity of DNA-A (<89% earlier, and <91% now) genome, different species have been identified. Two species namely Tomato leaf curl New Delhi virus and Tomato leaf curl Palampur virus predominantly distributed in Northern India and one species namely Tomato leaf curl Bangalore virus is dominant in Southern India [9, 14, 15].

The genus Begomovirus is a circular single-stranded DNA virus which is exclusively being transmitted by whitefly (Bemisia tabaci) in a persistent circulative manner [16]. Most of the begomovirus species are monopartite (having DNA-A molecule only) except few species, which are bipartite (having DNA-A and DNA-B as genomic component). However, in monopartite species, in addition to DNA-A molecule, betasatellite DNA is also present which is almost half of DNA-A component. The DNA-A of both mono- and bipartite species coding for six open reading frames (ORF), two in sense orientation namely AV1 and AV2 and four in complementary orientation namely AC1, AC2, AC3, and AC4, mainly are involved in virus replication and transmission. On the other hand, DNA-B of bipartite species coding for two ORF one in sense orientation namely BV1 and another in complementary orientation namely

primers or probes to virus species.

96 Advances in Plant Pathology

5. Begomovirus species and its genome organization

It has been reported earlier that tomato-infecting begomoviruses have bipartite genome in North India, while monopartite genome in South India [18]. Association of betasatellite DNA molecule with the ToLCD occurring in several places of India was reported [19].

In India, the population of tomato leaf curl viruses is so diverse, and it was shown after coat protein analysis of the 29 infected tomato samples. Based on these analysis, five clusters (with less than 88% similarity among them) were observed among the population, whereas four of them represented the tomato leaf curl viruses. Out of five, one cluster showed 89% similarity with Croton yellow vein mosaic virus [20]. As of now, so many closely related tomato-infecting begomovirus have been cloned and sequenced from India.

Figure 2. Genome organization of begomovirus associated with leaf curl disease of tomato.

## 6. Management

Management of viruses is difficult as viruses are systemic in nature, highly variable or diverse, insect vectors, and so on. No effective control measures of the ToLCVs associated with tomato have been developed so far for successful management except resistance, management of insect vectors, and altering the dates of sowing to avoid peaks of insect vector population. Management of ToLCV could be achieved by following various management practices.

and "Vaibhav") were developed and released officially in 2003–2004 in India. "Gene pyramiding" is combining multiple Ty genes in tomatoes with resistance to several whitefly-transmitted begomoviruses that cause TYLCVD [28]. Until now, six genes (Ty genes) derived from different tomato wild species have been identified. Prasanna et al. [22] attempted to combine Ty-2 and Ty-3 genes through marker-assisted selection and screened the hybrid lines for resistance to viruses by challenging through agroinoculation of specific, monopartite, and bipartite viruses and found that the lines and hybrids with Ty-2 were susceptible to ToLCNDV. The Ty-3 gene showed dosage effect with partial resistance of plants to ToLCNDV in the heterozygotes stage. By pyramiding Ty-2 and Ty-3 genes, considerable resistance to ToLCNDV can be achieved. The resistance of some of the tomato genotypes Vaibav, Nandhini, having Ty-2 gene to ToLCBaV were lost, when these genotypes were individually agroinoculated with ToLCBaV [13] and the cognate

Leaf Curl Disease: A Significant Constraint in the Production of Tomato in India

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h. Chemical control: foliar spray of neem (azadairachtin) and neem plus can kill the eggs, nymphs, and adults of B. tabaci. Ethanolic and aqueous extracts of Annona squamosal, Carlowrightia myriantha, Trichillia arborea, Azadirachta indica, and Acalypha gaumeri are effective against B. tabaci population. Neem oil, garlic, and eucalyptus extract give significant results against this disease [23]. The chemical control method is easy and most commonly used approach against the insect pest. A number of insecticides are used. Among them, imidacloprid, acetamiprid, nitenpyram, thiamethoxam, and diafenthiuron give significant

Begomoviruses are the most important viral pathogens in the Indian subcontinent. Tomato leaf curl disease is one of the devastating diseases and has been reported to be associated with several begomoviruses, thus making breeding for resistance more challenging. Adding to this, presence of diverse betasatellite increases complexity. Climate change and injudicious use of pesticides ensure the persistence of whitefly throughout the year and pose further challenges for the management of the disease. Besides the viral-induced symptom, symptoms caused by sucking of thrips and whitefly confuse the breeder and increase the difficulty for formulating management strategy. A thorough understanding on variability of the virus complexes and understanding the epidemiology could be an alternative in devising management strategy.

Division of Plant Pathology, Indian Agricultural Research Institute (IARI), New Delhi, India

results against aphids, whiteflies, and other insect pests [24, 25].

betasatellite.

7. Conclusion

Author details

Pradeep Kumar and Manish Kumar\*

\*Address all correspondence to: sharma90manish@gmail.com


and "Vaibhav") were developed and released officially in 2003–2004 in India. "Gene pyramiding" is combining multiple Ty genes in tomatoes with resistance to several whitefly-transmitted begomoviruses that cause TYLCVD [28]. Until now, six genes (Ty genes) derived from different tomato wild species have been identified. Prasanna et al. [22] attempted to combine Ty-2 and Ty-3 genes through marker-assisted selection and screened the hybrid lines for resistance to viruses by challenging through agroinoculation of specific, monopartite, and bipartite viruses and found that the lines and hybrids with Ty-2 were susceptible to ToLCNDV. The Ty-3 gene showed dosage effect with partial resistance of plants to ToLCNDV in the heterozygotes stage. By pyramiding Ty-2 and Ty-3 genes, considerable resistance to ToLCNDV can be achieved. The resistance of some of the tomato genotypes Vaibav, Nandhini, having Ty-2 gene to ToLCBaV were lost, when these genotypes were individually agroinoculated with ToLCBaV [13] and the cognate betasatellite.

h. Chemical control: foliar spray of neem (azadairachtin) and neem plus can kill the eggs, nymphs, and adults of B. tabaci. Ethanolic and aqueous extracts of Annona squamosal, Carlowrightia myriantha, Trichillia arborea, Azadirachta indica, and Acalypha gaumeri are effective against B. tabaci population. Neem oil, garlic, and eucalyptus extract give significant results against this disease [23]. The chemical control method is easy and most commonly used approach against the insect pest. A number of insecticides are used. Among them, imidacloprid, acetamiprid, nitenpyram, thiamethoxam, and diafenthiuron give significant results against aphids, whiteflies, and other insect pests [24, 25].

## 7. Conclusion

6. Management

98 Advances in Plant Pathology

transmission?

Management of viruses is difficult as viruses are systemic in nature, highly variable or diverse, insect vectors, and so on. No effective control measures of the ToLCVs associated with tomato have been developed so far for successful management except resistance, management of insect vectors, and altering the dates of sowing to avoid peaks of insect vector population. Management of ToLCV could be achieved by following various management practices.

a. Crop rotation can be utilized for management of disease spread by naturally breaking the life cycles of insect vectors, disease, and weeds. Rotating to nonhost crops prevents the buildup of large populations of the insect vector and also establishes host-free periods. b. Introducing a host-free period may delay infection in tomato by reduced whitefly population due to unavailability of proper host, which ultimately leads to lower rates of virus

c. Careful monitoring of sequential plantings should be done for virus management. Avoid sowing of tomato close to already infected fields. Synchronized planting should be

d. The source and use of crop transplants are also important in reducing or delaying infection. Early infection of susceptible seedlings should be monitored prior to transplanting. Nurseries should produce seedlings for commercial distribution in insectproof environment or under net cover to minimize infestation by the vector and subsequent virus transmission prior to transplanting. Roguing, or immediate removal of infected individual plants, may assist in delaying virus spread once the infected material

is immediately destroyed and not left to compost near adjacent, developing fields.

e. Reflective plastic mulches, yellow plastic mulch, and whitefly-proof screens can be

f. Biological control: biopesticides, a mass-produced agent manufactured from a living microorganism or a natural product, may offer a solution to disease control through introduction of predators and parasitoids of the vector. Biopesticides based on microbials such as Beauveria bassiana (effective on nymphs and adults) or Paecilomyces fumosoroseus, Green lacewings, ladybirds, minute pirate bugs, big-eyed bugs, and damsel bugs and Encarsia bimaculata [21] based on microbials such as Beauveria bassiana (effective on nymphs and adults) or Paecilomyces fumosoroseus. Green lacewings, ladybirds, minute pirate bugs, bigeyed bugs, damsel bugs and the parasitoid, Encarsia bimaculata [21]. Encarsia formosa is one of the most efficient and studied bioagents of B. tabaci. In a recent study, [27] has shown the biological control of ToLCV in tomato by application of chitosan-supplemented formulations of Pseudomonas sp. under field conditions. They also observed the higher levels of phenolics, phenylalanine ammonia lyase, peroxidase, and enhanced chitinase activity in

g. Host resistance: resistance approach is an easy, more effective approach for control of viral diseases. Three ToLCV-resistant open-pollinated tomato varieties ("Sankranthi," "Nandi"

followed to avoid initial inoculum from tomato plants.

employed to reduce the incidence of ToLCV-infected tomatoes.

rhizobacteria-treated plants (Mishra et al., 2014).

Begomoviruses are the most important viral pathogens in the Indian subcontinent. Tomato leaf curl disease is one of the devastating diseases and has been reported to be associated with several begomoviruses, thus making breeding for resistance more challenging. Adding to this, presence of diverse betasatellite increases complexity. Climate change and injudicious use of pesticides ensure the persistence of whitefly throughout the year and pose further challenges for the management of the disease. Besides the viral-induced symptom, symptoms caused by sucking of thrips and whitefly confuse the breeder and increase the difficulty for formulating management strategy. A thorough understanding on variability of the virus complexes and understanding the epidemiology could be an alternative in devising management strategy.

## Author details

Pradeep Kumar and Manish Kumar\*

\*Address all correspondence to: sharma90manish@gmail.com

Division of Plant Pathology, Indian Agricultural Research Institute (IARI), New Delhi, India

## References

[1] Butter NS, Rataul HS. Nature and extent of losses in tomatoes due to tomato leaf curl virus (TLCV) transmitted by whitefly, Bemisia tabaci Gen. (Hemiptera, Aleyrodidae). Indian Journal of Ecology. 1981;8:299-300

[15] Kirthi N, Maiya SP, Murthy MRN, Savithri HS. Evidence for recombination among the tomato leaf curl virus strains/species from Bangalore. India. Archives of Virology. 2002;

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[16] Rubinstein G, Czosnek H. Long-term association of tomato yellow leaf curl virus with its whitefly vector Bemisia tabaci: Effect on the insect transmission capacity, longevity and

[17] Rojas MR, Hagen C, Lucas WJ, Gilbertson RL. Exploiting chinks in the plant's armor: Evolution and emergence of geminiviruses. Annual Review of Phytopathology. 2005;43:

[18] Varma A, Malathi VG. Emerging geminivirus problems: A serious threat to crop produc-

[19] Sivalingam PN, Varma A. Role of betasatellite in the pathogenesis of a bipartite

[20] Chowda Reddy RV, Colvin J, Muniyappa V, Seal S. Diversity and distribution of bego-

[21] Qian M, Hu Q, Ren S, Mandour NS, Qiu B, Stansly PA. Delayed development of the whitefly (Bemisia tabaci) and increased parasitism by Encarsia bimaculata in response to sublethal doses

[22] Prasanna HC, Sinha DP, Rai GK, Krishna R, Kashyap SP, Singh NK, Malathi VG. Pyramiding Ty-2 and Ty-3 genes for resistance to monopartite and bipartite tomato leaf

[23] Khan MH, Ahmad N, Rashdi S, Rauf I, Ismail M, Tofique M. Management of sucking complex in bt cotton through the application of different plant products. Pakistan Journal

[24] Bacci LAL, Crespo TL, Galvan EJ, Pereira MC, Picanço GA, Chediak M. Toxicity of insecticides to the sweetpotato whitefly (Hemiptera: Aleyrodidae) and its natural ene-

[25] Mandal KA, Praveen KM, Subrata D, Arup C. Effective management of major tomato diseases in the gangetic plains of Eastern India through integrated approach. Agriculture Research & Technology. 2017;10(5):555796. DOI: 10.19080/ARTOAJ.2017.10.555796 [26] Mishra S, Jagadeesh KS, Krishnaraj PU, Prem S. Biocontrol of tomato leaf curl virus (ToLCV) in tomato with chitosan supplemented formulations of Pseudomonas sp. under

field conditions. Australian Journal of Crop Science. 2014;8(3):347-355

begomovirus affecting tomato in India. Archives of Virology. 2010;12:17-22

moviruses infecting tomato in India. Archives of Virology. 2005;150:845-867

of piperonyl butoxide. Insect Science. 2011. DOI: 10.1111/j.1744-7917.2011.01461.x

fecundity. Journal of General Virology. 1997;78:2683-2689

tion. The Annals of Applied Biology. 2003;142:145-164

curl viruses of India. Plant Pathology. 2015;64(2):256-264

mies. Pest Management Science. 2007;63(7):699-706

of Life Science. 2013;1(01):42-48

[27] National horticulture database. 2013 [28] http://avrdc.org/seed/improved-lines/

147:255-272

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Journal of Ecology. 1981;8:299-300

Journal of Mycology and Plant Pathology. 1973;3:50-54

Proceedings National Academy Science India. 1964;34:179-187

India. Tropical Agriculture. 1989;66:350-354

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virus. Plant Pathology. 2004;53:235

Virology. 1995;76:25-35

Varanasi, India. Phytopathology. 2003;93:1485-1495

and Management. IARI, New Delhi; February 19–21, (2004): 25

(1996): pp 229-234

260-262

[1] Butter NS, Rataul HS. Nature and extent of losses in tomatoes due to tomato leaf curl virus (TLCV) transmitted by whitefly, Bemisia tabaci Gen. (Hemiptera, Aleyrodidae). Indian

[2] Kalloo G. Leaf curl virus of tomato and chilli in India. In: Proceeding of the phase I final workshop of the South Asian Vegetable Research Network. Kathmandu; January 23–28,

[3] Sastry KSM, Singh SJ. Assessment of losses in tomato by tomato leaf curl virus. Indian

[4] Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X. Geminivirus

[5] Stanley J, Gay MR. Nucleotide sequence of cassava latent virus DNA. Nature. 1983;301:

[6] Vasudeva RS, Sam Raj J. A leaf curl disease of tomato. Phytopathology. 1948;38:364-369

[7] Singh DV, Lal SB. Occurrence of viruses of tomato and their probabales strains at Kanpur.

[8] Saikia AK, Muniyappa V. Epidemiology and control of tomato leaf curl virus in Southern

[9] Muniyappa V, Venkatesh HM, Ramappa HK, Kulkarni RS, Zeidan M, Tarba CY, Ghanim M, Croznek H. Tomato leaf curl virus from Bangalore (ToLCV-Ban4): Sequence comparison with Indian ToLCV isolates, detection in plants and insects and vector relationships.

[10] Chakraborty S, Pandey PK, Banerjee MK, Kalloo G, Fauguet CM. Tomato leaf curl Gujaratvirus, a new begomovirus species causing a severe leaf curl disease of tomato in

[11] Chatchawankaphanich O, Maxwell DP. Tomato leaf curl Karnataka virus from Bangalore, India, appears to be a recombinant Begomovirus. Phytopathology. 2002;12:637-645 [12] Sohrab SS, Mandal B, Varma A. Characterization of the virus associated with Lufa mosaic disease in northern India. National Symposium on Crop Surveillance: Disease Forecasting

[13] Usharani KS, Surendranath B, Paul-Khurana SM, Garg ID, Malathi VG. Potato leaf curlanew disease of potato in Northern India caused by a strain of tomato leaf curl New Delhi

[14] Padidam M, Beachy RN, Fauquet CM. Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. The Journal of General

strain demarcation and nomenclature. Archives of Virology. 2008;153(4):783-821

[28] http://avrdc.org/seed/improved-lines/

**Chapter 6**

**Provisional chapter**

**Plant Defense and Counter Defense by Viruses**

**Plant Defense and Counter Defense by Viruses**

DOI: 10.5772/intechopen.79114

RNA silencing is a robust sequence-specific RNA degradation process triggered by the formation of double-stranded RNA (dsRNA). RNA silencing was first discovered in transgenic plants, where it was termed co-suppression or post-transcriptional gene silencing (PTGS). In plants, it serves as an antiviral defense, and small RNA pathways serve as a defense against viruses and other invading nucleic acids. This chapter focuses on the interactions between host small RNA pathways and viral suppressors of silencing. Invading viruses carry genetic material that controls the host cell's machinery and tricks it into producing proteins and new viruses. Through RNA silencing, plant cells recognize this viral genetic material, remember and copy it so that other cells in the organism can be warned to destroy the virus. All cells in microbes, fungi, plants and mammals employ RNA silencing. However, viruses are known to fight back using RNA silencing suppressors, proteins that inhibit this defense mechanism. RNA silencing suppressors have been reported recently in other forms of pathogens like bacteria and oomycetes, which suggest that these pathogens have this inherent capability of counter defense across various kingdoms. In this chapter, we discuss some of these phenomenal counter defense mechanisms by the viruses.

**Keywords:** RNA silencing, PTGS, antiviral defense, silencing suppressors

As a matter of surprise, plant scientists, during the last decade of twentieth century, developed enthusiasm toward mechanism of gene silencing by virtue of plant transformation experiments, in which the introduction of a transgene into genome led to the silencing of transgene and homologous endogene [1, 2]. From these initial studies, plant biologists gained a wealth of information from gene silencing mechanisms and their complex pathways including their

> © 2016 The Author(s). Licensee InTech. This chapter is 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.

© 2018 The Author(s). Licensee IntechOpen. This chapter is 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.

Muzafar Ahmad Sheikh

Muzafar Ahmad Sheikh

**Abstract**

**1. Introduction**

**1.1. The beginning of a story**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79114

#### **Plant Defense and Counter Defense by Viruses Plant Defense and Counter Defense by Viruses**

DOI: 10.5772/intechopen.79114

#### Muzafar Ahmad Sheikh Muzafar Ahmad Sheikh

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79114

#### **Abstract**

RNA silencing is a robust sequence-specific RNA degradation process triggered by the formation of double-stranded RNA (dsRNA). RNA silencing was first discovered in transgenic plants, where it was termed co-suppression or post-transcriptional gene silencing (PTGS). In plants, it serves as an antiviral defense, and small RNA pathways serve as a defense against viruses and other invading nucleic acids. This chapter focuses on the interactions between host small RNA pathways and viral suppressors of silencing. Invading viruses carry genetic material that controls the host cell's machinery and tricks it into producing proteins and new viruses. Through RNA silencing, plant cells recognize this viral genetic material, remember and copy it so that other cells in the organism can be warned to destroy the virus. All cells in microbes, fungi, plants and mammals employ RNA silencing. However, viruses are known to fight back using RNA silencing suppressors, proteins that inhibit this defense mechanism. RNA silencing suppressors have been reported recently in other forms of pathogens like bacteria and oomycetes, which suggest that these pathogens have this inherent capability of counter defense across various kingdoms. In this chapter, we discuss some of these phenomenal counter defense mechanisms by the viruses.

**Keywords:** RNA silencing, PTGS, antiviral defense, silencing suppressors

#### **1. Introduction**

#### **1.1. The beginning of a story**

As a matter of surprise, plant scientists, during the last decade of twentieth century, developed enthusiasm toward mechanism of gene silencing by virtue of plant transformation experiments, in which the introduction of a transgene into genome led to the silencing of transgene and homologous endogene [1, 2]. From these initial studies, plant biologists gained a wealth of information from gene silencing mechanisms and their complex pathways including their

mutual multiserial interactions that they express. Besides, the biologists have made prominent achievements in using RNA silencing as a powerful tool for studying gene expression and crop improvements. RNA silencing (also called as posttranscriptional gene silencing PTGS) refers to a family of gene silencing effects by which the expression of one or more genes is downregulated or entirely suppressed by the introduction of the antisense RNA molecule. The most common and studied representation is RNA interference (RNAi). RNAi is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. It also plays a crucial role in defending plants against viruses. Enzymes search double-stranded RNA (dsRNA) that is normally present in cells and digest it into small pieces that render them inefficient to cause disease. The phenomenon of RNAi process can be divided into three steps. First, a long double-stranded RNA (dsRNA) that is introduced into the cell is modified into small RNA duplexes by a ribonuclease III (RNAase III) enzyme known as DICER; second, these duplexes are unwound and one of the strands is advantageously loaded into a protein complex known as the RNA-induced silencing complex, and at this point, RISC binds to an ARGONAUTE (AGO) protein. Third, this complex essentially scans the transcriptome and locates target RNAs. The packed ssRNA called the gRNA (guide RNA) directs an endonuclease that is present in RISC (also known as slicer), now known to be an Argonaute protein to split mRNAs that contain a sequence homologous to the siRNA. The importance of siRNAs in antiviral defense is to direct RISC complex to viral genomic and sub-genomic RNAs, thereby targeting those molecules for destruction (**Figure 1** Courtesy Trends in Microbiology, Vol.16. No.5). Multifarious use of siRNAs as specificity factors has been demonstrated in antiviral defense. The dsRNA sequence source for various RNA and DNA viruses is not known, but it is likely that they could have originated during viral

replication and/or from internal pairing of long RNA molecules [3]. RNA silencing is a highly complex system consisting of various proteins and processes [4]. This complexity makes the phenomenon of RNA silencing efficient for endogenous RNA expression during plant development and growth as well as for controlling viral infection. Viruses have adapted a robust mechanism for combating plant defense machinery by expressing suppressor proteins, which are capable of interfering in the process of RNA interference (RNAi) silencing pathway.

Plant Defense and Counter Defense by Viruses http://dx.doi.org/10.5772/intechopen.79114 105

• Double-stranded RNA (dsRNA) rather than single-stranded antisense RNA is the intrusive

• It circumvents the problems caused by knocked out genes in early stages (which could veil

• Silencing actions are passed through ages. It means that some of the dsRNA molecules could not silence the genes in parent, but the same ds RNA molecule did silence genes in

RNA silencing is predominantly a robust defense mechanism adapted by plants against pathogenic intruders especially viral pathogens. Viral pathogens have the ability to stay by suppressing RNA silencing. Plant viruses have developed tricky measures to overcome the host silencing response. One of these strategies is to overcome host-silencing response by producing proteins that target the signaling steps of RNA silencing [5]. Plants use RNA-silencing mechanism and produce short interfering RNA (SiRNA) molecules in a defense response against viral infection. To counter this defense response, virus produces suppressor proteins that can block the host silencing pathway or interfere with its function in plant cells [6]. The evidence of virus encoding RNA silencing suppressor proteins begins from experiments when silenced transgenes were again activated after virus inoculation. Silencing suppressors have been identified in DNAcontaining viruses and from positive-strand RNA viruses [6]. Collectively, the plant viral silencing suppressors are diverse in sequence and evolutionary origin. Being functionally diverse, they target cell autonomous steps and systemic signaling steps of RNA silencing mechanism. Before discussing the specific silencing suppressors, it is important to consider the consequences. At the earliest, there would appear to be a conflict, and later, many viruses encode the protein suppressors that block the signaling steps of RNA silencing. From the pioneer experiments of first viral suppressors of silencing in 1998, the scientists have generated a wealth of information about plant viral proteins that block silencing pathway elucidating their mechanism of action [7, 8]. Virus-encoded RNA-silencing suppressors interfere with various steps of the different silencing pathways and the mechanisms of suppression are being unraveled more and more.

the offspring because the dsRNA sequence did not match any of the parent genes.

• Silencing can be introduced in various progressive stages of the mechanism.

• RNAi is of high-degree specificity gene silencing mechanism.

Features of RNAi

desired observations).

**2. Viruses fight back**

agent.

**Figure 1.** RNA-cleavage activity leading to the degradation of AGO1.

replication and/or from internal pairing of long RNA molecules [3]. RNA silencing is a highly complex system consisting of various proteins and processes [4]. This complexity makes the phenomenon of RNA silencing efficient for endogenous RNA expression during plant development and growth as well as for controlling viral infection. Viruses have adapted a robust mechanism for combating plant defense machinery by expressing suppressor proteins, which are capable of interfering in the process of RNA interference (RNAi) silencing pathway.

Features of RNAi


## **2. Viruses fight back**

**Figure 1.** RNA-cleavage activity leading to the degradation of AGO1.

mutual multiserial interactions that they express. Besides, the biologists have made prominent achievements in using RNA silencing as a powerful tool for studying gene expression and crop improvements. RNA silencing (also called as posttranscriptional gene silencing PTGS) refers to a family of gene silencing effects by which the expression of one or more genes is downregulated or entirely suppressed by the introduction of the antisense RNA molecule. The most common and studied representation is RNA interference (RNAi). RNAi is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. It also plays a crucial role in defending plants against viruses. Enzymes search double-stranded RNA (dsRNA) that is normally present in cells and digest it into small pieces that render them inefficient to cause disease. The phenomenon of RNAi process can be divided into three steps. First, a long double-stranded RNA (dsRNA) that is introduced into the cell is modified into small RNA duplexes by a ribonuclease III (RNAase III) enzyme known as DICER; second, these duplexes are unwound and one of the strands is advantageously loaded into a protein complex known as the RNA-induced silencing complex, and at this point, RISC binds to an ARGONAUTE (AGO) protein. Third, this complex essentially scans the transcriptome and locates target RNAs. The packed ssRNA called the gRNA (guide RNA) directs an endonuclease that is present in RISC (also known as slicer), now known to be an Argonaute protein to split mRNAs that contain a sequence homologous to the siRNA. The importance of siRNAs in antiviral defense is to direct RISC complex to viral genomic and sub-genomic RNAs, thereby targeting those molecules for destruction (**Figure 1** Courtesy Trends in Microbiology, Vol.16. No.5). Multifarious use of siRNAs as specificity factors has been demonstrated in antiviral defense. The dsRNA sequence source for various RNA and DNA viruses is not known, but it is likely that they could have originated during viral

104 Advances in Plant Pathology

RNA silencing is predominantly a robust defense mechanism adapted by plants against pathogenic intruders especially viral pathogens. Viral pathogens have the ability to stay by suppressing RNA silencing. Plant viruses have developed tricky measures to overcome the host silencing response. One of these strategies is to overcome host-silencing response by producing proteins that target the signaling steps of RNA silencing [5]. Plants use RNA-silencing mechanism and produce short interfering RNA (SiRNA) molecules in a defense response against viral infection. To counter this defense response, virus produces suppressor proteins that can block the host silencing pathway or interfere with its function in plant cells [6]. The evidence of virus encoding RNA silencing suppressor proteins begins from experiments when silenced transgenes were again activated after virus inoculation. Silencing suppressors have been identified in DNAcontaining viruses and from positive-strand RNA viruses [6]. Collectively, the plant viral silencing suppressors are diverse in sequence and evolutionary origin. Being functionally diverse, they target cell autonomous steps and systemic signaling steps of RNA silencing mechanism. Before discussing the specific silencing suppressors, it is important to consider the consequences. At the earliest, there would appear to be a conflict, and later, many viruses encode the protein suppressors that block the signaling steps of RNA silencing. From the pioneer experiments of first viral suppressors of silencing in 1998, the scientists have generated a wealth of information about plant viral proteins that block silencing pathway elucidating their mechanism of action [7, 8]. Virus-encoded RNA-silencing suppressors interfere with various steps of the different silencing pathways and the mechanisms of suppression are being unraveled more and more.

#### **2.1. Suppressor and small RNA function**

The prodigy of accumulation of primary siRNAs to considerable level in the presence of some suppressors and that the target RNA degradation blockage indicates that primary siRNAs are not functional. These findings have suggested viral suppressors of RNA silencing bind to small RNA duplexes, after they bind properly to small RNA duplexes, they separate them and prevent their entry into RNA-induced silencing complex or RISC effector complex (**Figure 1**) [9, 10] This separation of small RNA duplexes has been suggested as a usual mode of action for RNA silencing suppression. The suppressors of RNA silencing can also change the biochemical structure of siRNAs, thereby blocking their function. Earlier findings have suggested that plant endogenous small RNA and transgene siRNAs have methylated group at their 3′ termini, this being an HUA ENHANCER<sup>1</sup> (HEN<sup>1</sup> ) relying process in their synthesis this step of methylation of viral siRNAs has been shown for DNA or RNA virus-infected plants. It has been demonstrated that many viruses and viral suppressors interfere with both siRNA and/ or miRNA methylation [11–13]. Moreover, the virus, alteration of host miRNA accumulation and function is believed to underlie at least some symptoms of plant virus infection [14, 15]. Despite the fact that maximum such research has focused on the role of viral suppressors, a recent study has shown that expression of other viral proteins can also affect miRNA accumulation and function [16]. Previous studies which considered plant viral suppressor's role in transgene induced silencing did not differentiate between primary and secondary siRNAs, and this led to topsy-turvies in the literature about whether a given suppressor did or did not block siRNA production. This ambiguity in results has been purposefully resolved, with the findings that some viral suppressors (i.e., P15 and P25) obstruct accumulation of primary siR-NAs, whereas other viral suppressors (i.e., P1/Hc-Pro, P39, P19 siRNAs) leave primary siRNA accumulation unimpaired, [10, 12, 17]. This specialized obstruction in secondary siRNA accumulation might be produced simply by suppressing primary siRNA function.

silencing await identification of the *Arabidopsis* homolog of the tobacco gene, which would open up numerous genetic approaches available in that model plant. Many viral protein suppressors of RNA silencing have been described so far, and extensive research was focused on

Plant Defense and Counter Defense by Viruses http://dx.doi.org/10.5772/intechopen.79114 107

The 2b protein of the cucumovirus was recognized as a silencing suppressor at about the same time as P1/HC-Pro of potyviruses. The CMV 2b protein, a nuclear protein that is required for long distance movement of the virus, functions as the silencing suppressor [20]. Viral-suppressor protein 2b interact directly with components of the RNA-induced silencing complex RISC machinery, 2b interact with AGO1, by inhibiting its RNA-cleavage activity leading to the degradation of Argonaute protein AGO1 (**Figure 1**). 2b specifically inhibits AGO1 cleavage activity in RISC reconstitution assessment. In addition, AGO1 recruit's virusderived small interfering RNAs (siRNAs) *in vivo*, suggesting that AGO1 is a major factor in defense against CMV infection. Viral suppressors of RNA silencing (VSRs) counter act RNAi based viral immunity. Many VSRs proteins have been reported, which play diverse functions in addition to suppressing RNA silencing, like viral replication, movement, coating and pathogenesis. Mostly plant viruses use VSRs as a tool to counter host defense machinery.

The P25 of the potexvirus, Potato virus X (PVX) is one of three cell-to-cell movement proteins (MPs) required for transport of virus from one cell to the next, the effects of P25 on cell autonomous and systemic silencing have been tested. Systemic silencing signal is a P25 – sensitive step and that the signal requires the transgene inducer pathway regardless of whether the inducer is a transgene or a replicating virus [14] However the fact that a viral protein inhibits the pathway leading to systemic signaling strongly implies that the systemic arm of the silencing response is part of the antiviral defense system. Studies have shown that the P25 protein encoded by potato virus X inhibits either the assembly or the function of the effector complexes of antiviral defense. Viruses counter the RNA silencing based counter defense by expressing VSRs. These VSRs are in turn recognized by host as avirulence (avr) factors to induce R- mediated resistance (Plant genomes carry many R genes that recognize specific

Helper-component proteinase (a pathogenicity regulator of potyviruses) is a necessary, multifunctional protein of the family *Potyviridae* initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced gene silencing, and the Hc-Pro protein product is required for suppression. Hc-Pro binds to ds siRNA intermediates and has been suggested to function by sequestering ds siRNAs or by inhibiting their unwinding to ss siRNAs [15, 21] **Figure 1** Courtesy Trends in Microbiology, Vol.16. No.5. HC-Pro in association with p25 or 2b targets intracellular and intercellular silencing respectively. This discovery regarding possible mechanism of silencing suppression was shown by interaction

a selection of these below mentioned proteins (suppressors).

**3.1. Cucumoviral 2b**

**3.2. Potexviral P25**

pathogens and induce resistance against them).

**3.3. Helper-component proteinase (HC-Pro)**

## **3. RNA silencing suppressors**

Plants defend themselves against viruses by RNA silencing; however, plant viruses spoil this defense machinery by expressing proteins that act as RNA silencing suppressors. Plants react to pathogens using elaborate networks of genetic interactions. Evidential progress has been made in understanding RNA silencing and how viruses counter this apparently ubiquitous antiviral defense. The best example of a viral suppressor that uses host factors that are not direct components of the silencing machinery to block silencing is the HC-Pro suppressor encoded by potyviruses. Two such factors have been reported so far [18, 19], and these will be discussed in more detail subsequently. The first is a calmodulin-like protein called regulator of gene silencing calmodulin-like (rgs-CaM). Tobacco rgs-CaM was identified as an HC-Prointeracting protein in a yeast two-hybrid screen, and subsequent experiments showed that overexpression of rgs-CaM interfered with virus-induced gene silencing (even in the absence of HC-Pro). Plants encode a large family of calmodulin-like proteins, which are characterized by the presence of a calmodulin domain with either amino-terminal or carboxy-terminal extensions. Experiments to determine if rgs-CaM is required for HC-Pro suppression of silencing await identification of the *Arabidopsis* homolog of the tobacco gene, which would open up numerous genetic approaches available in that model plant. Many viral protein suppressors of RNA silencing have been described so far, and extensive research was focused on a selection of these below mentioned proteins (suppressors).

## **3.1. Cucumoviral 2b**

**2.1. Suppressor and small RNA function**

106 Advances in Plant Pathology

termini, this being an HUA ENHANCER<sup>1</sup>

**3. RNA silencing suppressors**

The prodigy of accumulation of primary siRNAs to considerable level in the presence of some suppressors and that the target RNA degradation blockage indicates that primary siRNAs are not functional. These findings have suggested viral suppressors of RNA silencing bind to small RNA duplexes, after they bind properly to small RNA duplexes, they separate them and prevent their entry into RNA-induced silencing complex or RISC effector complex (**Figure 1**) [9, 10] This separation of small RNA duplexes has been suggested as a usual mode of action for RNA silencing suppression. The suppressors of RNA silencing can also change the biochemical structure of siRNAs, thereby blocking their function. Earlier findings have suggested that plant endogenous small RNA and transgene siRNAs have methylated group at their 3′

(HEN<sup>1</sup>

mulation might be produced simply by suppressing primary siRNA function.

Plants defend themselves against viruses by RNA silencing; however, plant viruses spoil this defense machinery by expressing proteins that act as RNA silencing suppressors. Plants react to pathogens using elaborate networks of genetic interactions. Evidential progress has been made in understanding RNA silencing and how viruses counter this apparently ubiquitous antiviral defense. The best example of a viral suppressor that uses host factors that are not direct components of the silencing machinery to block silencing is the HC-Pro suppressor encoded by potyviruses. Two such factors have been reported so far [18, 19], and these will be discussed in more detail subsequently. The first is a calmodulin-like protein called regulator of gene silencing calmodulin-like (rgs-CaM). Tobacco rgs-CaM was identified as an HC-Prointeracting protein in a yeast two-hybrid screen, and subsequent experiments showed that overexpression of rgs-CaM interfered with virus-induced gene silencing (even in the absence of HC-Pro). Plants encode a large family of calmodulin-like proteins, which are characterized by the presence of a calmodulin domain with either amino-terminal or carboxy-terminal extensions. Experiments to determine if rgs-CaM is required for HC-Pro suppression of

of methylation of viral siRNAs has been shown for DNA or RNA virus-infected plants. It has been demonstrated that many viruses and viral suppressors interfere with both siRNA and/ or miRNA methylation [11–13]. Moreover, the virus, alteration of host miRNA accumulation and function is believed to underlie at least some symptoms of plant virus infection [14, 15]. Despite the fact that maximum such research has focused on the role of viral suppressors, a recent study has shown that expression of other viral proteins can also affect miRNA accumulation and function [16]. Previous studies which considered plant viral suppressor's role in transgene induced silencing did not differentiate between primary and secondary siRNAs, and this led to topsy-turvies in the literature about whether a given suppressor did or did not block siRNA production. This ambiguity in results has been purposefully resolved, with the findings that some viral suppressors (i.e., P15 and P25) obstruct accumulation of primary siR-NAs, whereas other viral suppressors (i.e., P1/Hc-Pro, P39, P19 siRNAs) leave primary siRNA accumulation unimpaired, [10, 12, 17]. This specialized obstruction in secondary siRNA accu-

) relying process in their synthesis this step

The 2b protein of the cucumovirus was recognized as a silencing suppressor at about the same time as P1/HC-Pro of potyviruses. The CMV 2b protein, a nuclear protein that is required for long distance movement of the virus, functions as the silencing suppressor [20]. Viral-suppressor protein 2b interact directly with components of the RNA-induced silencing complex RISC machinery, 2b interact with AGO1, by inhibiting its RNA-cleavage activity leading to the degradation of Argonaute protein AGO1 (**Figure 1**). 2b specifically inhibits AGO1 cleavage activity in RISC reconstitution assessment. In addition, AGO1 recruit's virusderived small interfering RNAs (siRNAs) *in vivo*, suggesting that AGO1 is a major factor in defense against CMV infection. Viral suppressors of RNA silencing (VSRs) counter act RNAi based viral immunity. Many VSRs proteins have been reported, which play diverse functions in addition to suppressing RNA silencing, like viral replication, movement, coating and pathogenesis. Mostly plant viruses use VSRs as a tool to counter host defense machinery.

### **3.2. Potexviral P25**

The P25 of the potexvirus, Potato virus X (PVX) is one of three cell-to-cell movement proteins (MPs) required for transport of virus from one cell to the next, the effects of P25 on cell autonomous and systemic silencing have been tested. Systemic silencing signal is a P25 – sensitive step and that the signal requires the transgene inducer pathway regardless of whether the inducer is a transgene or a replicating virus [14] However the fact that a viral protein inhibits the pathway leading to systemic signaling strongly implies that the systemic arm of the silencing response is part of the antiviral defense system. Studies have shown that the P25 protein encoded by potato virus X inhibits either the assembly or the function of the effector complexes of antiviral defense. Viruses counter the RNA silencing based counter defense by expressing VSRs. These VSRs are in turn recognized by host as avirulence (avr) factors to induce R- mediated resistance (Plant genomes carry many R genes that recognize specific pathogens and induce resistance against them).

#### **3.3. Helper-component proteinase (HC-Pro)**

Helper-component proteinase (a pathogenicity regulator of potyviruses) is a necessary, multifunctional protein of the family *Potyviridae* initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced gene silencing, and the Hc-Pro protein product is required for suppression. Hc-Pro binds to ds siRNA intermediates and has been suggested to function by sequestering ds siRNAs or by inhibiting their unwinding to ss siRNAs [15, 21] **Figure 1** Courtesy Trends in Microbiology, Vol.16. No.5. HC-Pro in association with p25 or 2b targets intracellular and intercellular silencing respectively. This discovery regarding possible mechanism of silencing suppression was shown by interaction between P1/HC-Pro of TEV (Tobacco etch virus) and rgs-CaM a tobacco calmodulin like protein [18]. It was demonstrated that rgs-CaM suppresses itself RNA silencing mechanism upon overexpression in the plants points to the role of gene silencing as a natural antiviral defense system in plants and offer different approaches to explain the molecular basis of gene silencing.

viruses, plants serve as natural hosts) suppress the plant's RNA silencing machinery. Here authors [28–30] identified a silencing suppressor protein (SSP), P0PE, in the genus *Enamovirus* with only one species *Pea enation mosaic virus*-1 (PEMV-1) and showed that it and the P0s of the Polerovirus *Potato leaf roll virus* and *Cereal yellow dwarf virus* have strong local and systemic SSP activity, while the P1 of genus *Sobemovirus* type species *Southern bean mosaic virus* suppresses systemic silencing. The nuclear localized P0PE has no observable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from the Polerovirus P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the Poleroviral and Enamoviral

Plant Defense and Counter Defense by Viruses http://dx.doi.org/10.5772/intechopen.79114 109

RNA silencing suppressors (RSSs) are very important factors for virus biology. Virus encodes RSS irrespective of their genome size, for example the geminivirus genome with only 2.7 kb genome size encodes three suppressors (AL2/AC2, AC4 and possibly βC1), whereas CTV with large genome size of 40 kb encodes three RSS. Therefore, the size of the genome is not an indicator of number RSS. Through their evolution, plants and pathogens have adapted and evolved a wide variety of sophisticated strategies to attack, defend, and counterattack. Plants have acquired abilities to sense and defend against invading pathogens by utilizing preexisting and/or induced barriers to stop infection. In parallel, plant pathogens have evolved diverse ways to counter or overcome host disease resistance. One of the common pathogen strategies involves the production of plant defense suppressors. Viruses evolve rapidly to their host organism and adapt themselves. In contrast, the cellular organisms evolve and adapt to a lesser extent. Keeping in view the fact that viruses possess antiviral defense system suggests that viruses and their host have coevolved. This interdependence among the life forms cannot be fully understood except in an evolutionary frame work. RNAi is a resistance defensive mechanism in plants which targets viral genomes and transcripts to degradation, several findings have revealed viral suppressors that target plant proteins and the possible actions that viruses take during their interference with the defense systems of the host: there remain many unanswered questions for example, the type of proteolysis machinery used by P0 to degrade its plant interactor AGO1 is a matter of debate and the mechanism by which V2 disrupts the RNAi- silencing system of the plant is unknown. The more we dig into the ongoing battle between viruses and their hosts, the more we come to know about the intriguing defense and counter-defense strategies that enable plants and viruses to coexist. To conclude, it can be stated that the interactions between antiviral RNA silencing and the counter measures viruses have evolved to frustrate such process is a continuously evolving action in the continuously evolving microbial world. On the one hand, a very important topic in virology, and on the other hand, a strong starting point for breakthroughs in other fields of research such as functional genomics and development. In an application environment, RNA silencing has allowed us to develop efficient and broad virus resistance in plants, which plays a crucial role to the reliable production of food. RNAi suppression holds the potential of unearthing many unexpected surprises and this promising field is the object of intense investigation.

SSPs have a conserved mode of action on the RNA silencing mechanism.

**4. Conclusion**

### **3.4. Tombusvirus P19**

From the time of its discovery, the Tombusvirus encoded P19 protein (P19) in the late 1980s, the status of this potent suppressor changed from being thought obsolete to its identification a decade later as an important viral pathogenicity factor. A recent study also has confirmed that *Pothos latent virus* (PLV) encode p14 silencing suppressor, although the genome of Tombusvirus is similar to PLV, its suppressor (p14) is smaller than P19 with higher affinity to long dsRNAs. Tombusvirus P19 is a protein encoded by *Tomato bushy stunt virus* and related tombusvirus. Studies have demonstrated that P19 and p14 are RNA silencing suppressors (RSS) in plant cells [22]. P19 was reported to suppress PTGS mainly along the vein tissue and in newly emerging leaves, whereas HC-Pro reversed PTGS in a non-tissue- specific manner [10, 23]. A study confirmed [10, 24] that


### **3.5. V2 suppressor**

The suppression of silencing is a key mechanism for successful viral entry The V2 protein of *Tomato yellow leaf curl China virus* (TYLCCNV) was identified as an RNA silencing suppressor by *Agrobacterium*-mediated co-infiltration. The V2 protein could inhibit local RNA silencing [25].

V2 suppressor of *Tomato yellow mosaic virus* binds the coiled-coil protein suppressor of the gene-silencing SGS3 homolog. These reports provide novel insight into the mechanisms developed by viruses to target the defense system of the plant [26]. DNA viruses from the Geminiviridae family encode several proteins namely C2, C4 and V2 which suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the potent PTGS suppressor. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among Geminiviruses. In this genus, C2/L2 protein has been described as inhibiting post-transcriptional gene silencing [27].

#### **3.6. P0 protein**

The P0 protein of the Polerovirus (Polerovirus is a genus of viruses, in the family Luteoviridae, plants serve as natural hosts) and P1 protein of the Sobemovirus (Sobemovirus is a genus of viruses, plants serve as natural hosts) suppress the plant's RNA silencing machinery. Here authors [28–30] identified a silencing suppressor protein (SSP), P0PE, in the genus *Enamovirus* with only one species *Pea enation mosaic virus*-1 (PEMV-1) and showed that it and the P0s of the Polerovirus *Potato leaf roll virus* and *Cereal yellow dwarf virus* have strong local and systemic SSP activity, while the P1 of genus *Sobemovirus* type species *Southern bean mosaic virus* suppresses systemic silencing. The nuclear localized P0PE has no observable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from the Polerovirus P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the Poleroviral and Enamoviral SSPs have a conserved mode of action on the RNA silencing mechanism.

## **4. Conclusion**

between P1/HC-Pro of TEV (Tobacco etch virus) and rgs-CaM a tobacco calmodulin like protein [18]. It was demonstrated that rgs-CaM suppresses itself RNA silencing mechanism upon overexpression in the plants points to the role of gene silencing as a natural antiviral defense system

From the time of its discovery, the Tombusvirus encoded P19 protein (P19) in the late 1980s, the status of this potent suppressor changed from being thought obsolete to its identification a decade later as an important viral pathogenicity factor. A recent study also has confirmed that *Pothos latent virus* (PLV) encode p14 silencing suppressor, although the genome of Tombusvirus is similar to PLV, its suppressor (p14) is smaller than P19 with higher affinity to long dsRNAs. Tombusvirus P19 is a protein encoded by *Tomato bushy stunt virus* and related tombusvirus. Studies have demonstrated that P19 and p14 are RNA silencing suppressors (RSS) in plant cells [22]. P19 was reported to suppress PTGS mainly along the vein tissue and in newly emerging leaves, whereas HC-Pro reversed PTGS in a non-tissue- specific manner

• P19 is a suppressor of viral induced gene silencing (VIGS), P19 can also fetter to ds siRNA by inhibiting their untwining to ss siRNAs, thereby counter the silencing mechanism.

The suppression of silencing is a key mechanism for successful viral entry The V2 protein of *Tomato yellow leaf curl China virus* (TYLCCNV) was identified as an RNA silencing suppressor by *Agrobacterium*-mediated co-infiltration. The V2 protein could inhibit local RNA silencing [25]. V2 suppressor of *Tomato yellow mosaic virus* binds the coiled-coil protein suppressor of the gene-silencing SGS3 homolog. These reports provide novel insight into the mechanisms developed by viruses to target the defense system of the plant [26]. DNA viruses from the Geminiviridae family encode several proteins namely C2, C4 and V2 which suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the potent PTGS suppressor. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among Geminiviruses. In this genus, C2/L2 protein has been described as

The P0 protein of the Polerovirus (Polerovirus is a genus of viruses, in the family Luteoviridae, plants serve as natural hosts) and P1 protein of the Sobemovirus (Sobemovirus is a genus of

in plants and offer different approaches to explain the molecular basis of gene silencing.

**3.4. Tombusvirus P19**

108 Advances in Plant Pathology

[10, 23]. A study confirmed [10, 24] that

• P19 is a potent suppressor of PTGS

inhibiting post-transcriptional gene silencing [27].

**3.5. V2 suppressor**

**3.6. P0 protein**

• P14 binds to long and short dsRNA including the siRNA duplex.

RNA silencing suppressors (RSSs) are very important factors for virus biology. Virus encodes RSS irrespective of their genome size, for example the geminivirus genome with only 2.7 kb genome size encodes three suppressors (AL2/AC2, AC4 and possibly βC1), whereas CTV with large genome size of 40 kb encodes three RSS. Therefore, the size of the genome is not an indicator of number RSS. Through their evolution, plants and pathogens have adapted and evolved a wide variety of sophisticated strategies to attack, defend, and counterattack. Plants have acquired abilities to sense and defend against invading pathogens by utilizing preexisting and/or induced barriers to stop infection. In parallel, plant pathogens have evolved diverse ways to counter or overcome host disease resistance. One of the common pathogen strategies involves the production of plant defense suppressors. Viruses evolve rapidly to their host organism and adapt themselves. In contrast, the cellular organisms evolve and adapt to a lesser extent. Keeping in view the fact that viruses possess antiviral defense system suggests that viruses and their host have coevolved. This interdependence among the life forms cannot be fully understood except in an evolutionary frame work. RNAi is a resistance defensive mechanism in plants which targets viral genomes and transcripts to degradation, several findings have revealed viral suppressors that target plant proteins and the possible actions that viruses take during their interference with the defense systems of the host: there remain many unanswered questions for example, the type of proteolysis machinery used by P0 to degrade its plant interactor AGO1 is a matter of debate and the mechanism by which V2 disrupts the RNAi- silencing system of the plant is unknown. The more we dig into the ongoing battle between viruses and their hosts, the more we come to know about the intriguing defense and counter-defense strategies that enable plants and viruses to coexist. To conclude, it can be stated that the interactions between antiviral RNA silencing and the counter measures viruses have evolved to frustrate such process is a continuously evolving action in the continuously evolving microbial world. On the one hand, a very important topic in virology, and on the other hand, a strong starting point for breakthroughs in other fields of research such as functional genomics and development. In an application environment, RNA silencing has allowed us to develop efficient and broad virus resistance in plants, which plays a crucial role to the reliable production of food. RNAi suppression holds the potential of unearthing many unexpected surprises and this promising field is the object of intense investigation.
