**7.** *Citrus tristeza virus* **genetics and plant-host interactions**

**Figure 5.** Different species of D-RNAs in CTV populations. Top panel: graphic representation of a usual small CTV D-RNA compared with the CTV genome. Bottom panel: accumulation of CTV RNAs in bark extracts from sweet orange plants infected with three field isolates (1 to 3 lines), before (lines w) and after (lines T) aphid transmission. Northernblot hybridization performed using a single-stranded minus-sense riboprobes specific to the 3' end (left panel) and

In other viral pathosystems, D-RNAs have the capacity of interfering with the viral replication process of their helper virus (named defective interfering (DI) RNAs), but those function was not reported for CTV [10]. Although their biological role is presently unknown, at least in one case, the presence of D-RNAs was suggested to modulate symptom development either increasing or decreasing CTV symptom expression [57]. Most of the CTV D-RNAs contain a complete region p23 and the 3´NTR [43] that is associated with SY development [20], thus they could have a role in symptom modulation. Therefore, it will be necessary to promote further research to elucidate the role of the D-RNAs (or DI-RNAs) in CTV replication or in modulation

the 5´end (right panel) of the T36 genome. From Mawassi et al. [46] and Albiach-Marti et al. [45]

10 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

of pathogenic responses in the infected plant host.

In order to infect a plant, CTV needs to enter in the cell, and to overcome the constitutive and/or inducible plant defences, to re-program the plant cellular machinery for its viral multiplica‐ tion. The infection process will continue with the assembly of new viral particles that will move celltocellthroughtheplasmodesmata.Thisprocesswillbecompletedwiththevirallongdistance movement through the plant vascular structure to colonize systemically the plant. Each CTV gene product seems to have a primary genetic function required for the survival of the virus. However, there are secondary genetic interactions, which cause or trigger resistance or pathogenic responses in the citrus host [60, 61]. *Citrus* genus contains multitude of species, cultivars and intergenic hybrids, with which CTV could interact causing a range of physiologi‐ cal and biochemical responses. These could be from either pathogenic or asymptomatic phenotypes to limited or complete plant resistance [5]. Although most of these mechanisms are still a mystery, new discoveries towards the understanding of the genetics of CTV movement in the plant, host-range, host resistance and pathogenicity have been reported recently [19, 20, 21, 62, 63, 64].

suppressors of the plant RNA silencing mechanism in *N. benthamiana* and *N. tabacum* plants. The p23 inhibits intercellular RNA silencing, while CP impedes intracellular RNA silencing and p20 limits both inter and intracellular RNA silencing [18]. Although, CP, p20 and p23 have not been yet reported as suppressors of the citrus silencing mechanism, their presence in the CTV genome is in concordance with wide host range among citrus species and hybrids, previously described, and consequently, with the complexity of CTV-citrus interactions. In fact, in spite of the existence of these three silencing suppressors, accumulation of siRNAs in CTV-infected susceptible hosts is 50% of the total RNAs in the plant [64]. The CTV siRNAs accumulation in infected plants is directly proportional to the virus accumulation and varies depending on the citrus host. Deep sequencing analysis of these siRNAs, from CTV-infected plants, indicated that they mainly consisted in small RNAs of 21-22 nt derived essentially from

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 13

**7.3. Genetic determinants of the** *Citrus tristeza virus* **pathogenic syndromes**

Viruses possess the potential to disrupt host physiology either by usurpation of substantial amount of plant metabolic resources or by the interaction of a specific viral product with the host components [60]. CTV induces three hallmark syndromes, plus a myriad different symptom patterns in indexing plants. *Tristeza* disease or QD syndrome consists in overgrowth of the scion at the bud union, loss of root mass, and therefore death of citrus commercial varieties grafted on sour orange rootstock [5]. The SP syndrome consists in deep pits in the wood under depressed areas of bark in commercial varieties of sweet orange and grapefruit trees grafted on any rootstock. Usually SP do not cause tree death, but severe stunting and unmarketable fruit, thus causing elevated economic damages [5]. The SY syndrome is characterized by stunting, leaf chlorosis and sometimes a complete cessation of growth on sour orange, grapefruit or lemon [*C. limon* (L.) Burm. f.] seedlings (Figure 6). Although, SY syn‐ drome might be found at the field in top–grafted plants and it is not economically valuable, it could be examined in the greenhouse in a timely manner and has a substantial diagnostic value for CTV pathotype differentiation [5]. On the contrary, the development of QD and SP extends over 10 to 40 years at the field [1], a period too long to screen the CTV isolates. Although SP pathotype could be likely examined in glasshouse conditions, there are no reliable methods to reproduce the QD in those conditions [5]. Therefore, the degree of severity of a specific CTV isolate, strain or genotype usually is assessed by using indexing plants (Mexican lime, *C. macrophylla*, sour orange, sweet orange and Duncan grapefruit) [60]. In this case, the degree of CTV symptomology ranges from the mild phenotypes, which are almost asymptomatic, to the highly virulent CTV isolates that could generate vein clearing, leaf cupping, dwarfing, stem pitting and the plant death [5]. This diversity and grade of symptom responses to CTV infection suggests the possible presence of more than one mechanism of pathogenicity taking place

CTV multiplication generates great quantities of viral products like, at least, 19 viral proteins, 35 RNA species (gRNAs, sgRNAs and LMTs) and D-RNAs along with a complicated process of replication, gene expression, assembly and movement, where the interaction with host factors is essential. Consequently, during the CTV-*Citrus* interaction there are multiple opportunities to generate disease. In fact, analysis of Mexican lime transcriptome using microarrays, after infection with a severe CTV isolate, showed altered expression of 334 genes

the CTV genome [64].

during the CTV-*Citrus* interactions.

## **7.1.** *Citrus tristeza virus* **host range and plant systemic infection**

*Citrus tristeza virus* natural plant hosts belong to the order *Geraniales*, family *Rutaceae*, subfamily *Aurantoidea*. There are also non-rutaceous hosts that have been experimentally infected with CTV strains like *Passiflora gracilis* or *Passiflora coerulea*. Some citrus hosts are usually susceptible to CTV infection like Mexican lime or *C. macrophylla*. Other citrus host are tolerant to some CTV strains like sweet orange [*C. sinensis* (L.) Osb.] and grapefruit (*C. paradisi* Macf.), or tolerant to almost all known CTV strains as mandarins (*C. reticulata* Blanco). Finally, pummelos [*C. grandis* (L.) Osb.], sour orange (*C. aurantium* L.) and the hybrid rootstock Swingle citrumelo exhibit a differential degree of resistance depending on the CTV strain. In addition, some *Citrus* relatives within subfamily *Aurantioideae,* like *Poncirus trifoliata* (L.) Raf., as well as *P. trifoliata* intergenic hybrids remain resistant or immune to most of the CTV strains [4, 5]. Consequently, these data highlight an elevated complexity in the CTV systemic infection and host range genetics.

Several CTV genes are related with systemic infection of citrus plants [14, 62]. Viral mu‐ tants with a deletion in the p6 and p20 ORFs failed to infect citrus plants systemically, suggesting their possible roles in virus translocation or infection of the whole plant. Like‐ wise, the p6 homologue in BYV is a movement protein [65], and similarly to homologous proteins function in BYV [11], CP, CPm, HSP70h and p61 probably participate in the viral movement. CTV genome has several ORFs that are non-conserved in the family *Closteroviri‐ dae,* thus unique for CTV. Unexpectedly, three of these ORFs (p33, p18 and p13) neither are required for replication and assembly [6, 13] nor for systemic infection of Mexican lime and *C. macrophylla* [14]. However, p33, p18 and p13 were demonstrated to be CTV host range determinants. The p33 ORF is necessary for the systemic infection of sour orange and lemon trees. Likewise, either p33 or p18 ORF is enough for systemic infection of grapefruit trees. Similarly, p33 or p13 ORF is sufficient to invade whole calamondin (*C. mitis*) plants. As a result of the acquisition of multiple non-conserved genes (p33, p18, and p13), probably CTV increased the possibilities to interact with multiple hosts, thus extending its host range during the course of its evolution [19].

#### **7.2.** *Citrus tristeza virus* **suppressing genes of plant silencing mechanism**

The plant constitutive defence consists of the RNA mediated post-transcriptional silencing mechanism (PTGS) that implies the specific degradation of the viral dsRNA in small interfering RNAs (siRNAs), which guides a specific plant ribonuclease to disintegrate the viral genomes in the cytoplasm. Besides the antiviral role, the plant silencing mechanism has important functions in regulating plant gene expression (miRNA metabolism) [60]. In order to infect plants, viruses developed a strategy to block this silencing mechanism: the suppressing genes. This strategy allows viral replication but interfere with host gene expression, thus inducing disease [60, 66]. As indicated previously, CTV evolved ending up with three proteins that are suppressors of the plant RNA silencing mechanism in *N. benthamiana* and *N. tabacum* plants. The p23 inhibits intercellular RNA silencing, while CP impedes intracellular RNA silencing and p20 limits both inter and intracellular RNA silencing [18]. Although, CP, p20 and p23 have not been yet reported as suppressors of the citrus silencing mechanism, their presence in the CTV genome is in concordance with wide host range among citrus species and hybrids, previously described, and consequently, with the complexity of CTV-citrus interactions. In fact, in spite of the existence of these three silencing suppressors, accumulation of siRNAs in CTV-infected susceptible hosts is 50% of the total RNAs in the plant [64]. The CTV siRNAs accumulation in infected plants is directly proportional to the virus accumulation and varies depending on the citrus host. Deep sequencing analysis of these siRNAs, from CTV-infected plants, indicated that they mainly consisted in small RNAs of 21-22 nt derived essentially from the CTV genome [64].

#### **7.3. Genetic determinants of the** *Citrus tristeza virus* **pathogenic syndromes**

still a mystery, new discoveries towards the understanding of the genetics of CTV movement in the plant, host-range, host resistance and pathogenicity have been reported recently [19, 20, 21,

*Citrus tristeza virus* natural plant hosts belong to the order *Geraniales*, family *Rutaceae*, subfamily *Aurantoidea*. There are also non-rutaceous hosts that have been experimentally infected with CTV strains like *Passiflora gracilis* or *Passiflora coerulea*. Some citrus hosts are usually susceptible to CTV infection like Mexican lime or *C. macrophylla*. Other citrus host are tolerant to some CTV strains like sweet orange [*C. sinensis* (L.) Osb.] and grapefruit (*C. paradisi* Macf.), or tolerant to almost all known CTV strains as mandarins (*C. reticulata* Blanco). Finally, pummelos [*C. grandis* (L.) Osb.], sour orange (*C. aurantium* L.) and the hybrid rootstock Swingle citrumelo exhibit a differential degree of resistance depending on the CTV strain. In addition, some *Citrus* relatives within subfamily *Aurantioideae,* like *Poncirus trifoliata* (L.) Raf., as well as *P. trifoliata* intergenic hybrids remain resistant or immune to most of the CTV strains [4, 5]. Consequently, these data highlight an elevated complexity in the CTV systemic infection

Several CTV genes are related with systemic infection of citrus plants [14, 62]. Viral mu‐ tants with a deletion in the p6 and p20 ORFs failed to infect citrus plants systemically, suggesting their possible roles in virus translocation or infection of the whole plant. Like‐ wise, the p6 homologue in BYV is a movement protein [65], and similarly to homologous proteins function in BYV [11], CP, CPm, HSP70h and p61 probably participate in the viral movement. CTV genome has several ORFs that are non-conserved in the family *Closteroviri‐ dae,* thus unique for CTV. Unexpectedly, three of these ORFs (p33, p18 and p13) neither are required for replication and assembly [6, 13] nor for systemic infection of Mexican lime and *C. macrophylla* [14]. However, p33, p18 and p13 were demonstrated to be CTV host range determinants. The p33 ORF is necessary for the systemic infection of sour orange and lemon trees. Likewise, either p33 or p18 ORF is enough for systemic infection of grapefruit trees. Similarly, p33 or p13 ORF is sufficient to invade whole calamondin (*C. mitis*) plants. As a result of the acquisition of multiple non-conserved genes (p33, p18, and p13), probably CTV increased the possibilities to interact with multiple hosts, thus extending its host range during

**7.2.** *Citrus tristeza virus* **suppressing genes of plant silencing mechanism**

The plant constitutive defence consists of the RNA mediated post-transcriptional silencing mechanism (PTGS) that implies the specific degradation of the viral dsRNA in small interfering RNAs (siRNAs), which guides a specific plant ribonuclease to disintegrate the viral genomes in the cytoplasm. Besides the antiviral role, the plant silencing mechanism has important functions in regulating plant gene expression (miRNA metabolism) [60]. In order to infect plants, viruses developed a strategy to block this silencing mechanism: the suppressing genes. This strategy allows viral replication but interfere with host gene expression, thus inducing disease [60, 66]. As indicated previously, CTV evolved ending up with three proteins that are

**7.1.** *Citrus tristeza virus* **host range and plant systemic infection**

12 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

62, 63, 64].

and host range genetics.

the course of its evolution [19].

Viruses possess the potential to disrupt host physiology either by usurpation of substantial amount of plant metabolic resources or by the interaction of a specific viral product with the host components [60]. CTV induces three hallmark syndromes, plus a myriad different symptom patterns in indexing plants. *Tristeza* disease or QD syndrome consists in overgrowth of the scion at the bud union, loss of root mass, and therefore death of citrus commercial varieties grafted on sour orange rootstock [5]. The SP syndrome consists in deep pits in the wood under depressed areas of bark in commercial varieties of sweet orange and grapefruit trees grafted on any rootstock. Usually SP do not cause tree death, but severe stunting and unmarketable fruit, thus causing elevated economic damages [5]. The SY syndrome is characterized by stunting, leaf chlorosis and sometimes a complete cessation of growth on sour orange, grapefruit or lemon [*C. limon* (L.) Burm. f.] seedlings (Figure 6). Although, SY syn‐ drome might be found at the field in top–grafted plants and it is not economically valuable, it could be examined in the greenhouse in a timely manner and has a substantial diagnostic value for CTV pathotype differentiation [5]. On the contrary, the development of QD and SP extends over 10 to 40 years at the field [1], a period too long to screen the CTV isolates. Although SP pathotype could be likely examined in glasshouse conditions, there are no reliable methods to reproduce the QD in those conditions [5]. Therefore, the degree of severity of a specific CTV isolate, strain or genotype usually is assessed by using indexing plants (Mexican lime, *C. macrophylla*, sour orange, sweet orange and Duncan grapefruit) [60]. In this case, the degree of CTV symptomology ranges from the mild phenotypes, which are almost asymptomatic, to the highly virulent CTV isolates that could generate vein clearing, leaf cupping, dwarfing, stem pitting and the plant death [5]. This diversity and grade of symptom responses to CTV infection suggests the possible presence of more than one mechanism of pathogenicity taking place during the CTV-*Citrus* interactions.

CTV multiplication generates great quantities of viral products like, at least, 19 viral proteins, 35 RNA species (gRNAs, sgRNAs and LMTs) and D-RNAs along with a complicated process of replication, gene expression, assembly and movement, where the interaction with host factors is essential. Consequently, during the CTV-*Citrus* interaction there are multiple opportunities to generate disease. In fact, analysis of Mexican lime transcriptome using microarrays, after infection with a severe CTV isolate, showed altered expression of 334 genes and about half of them without significant similarity with other known sequences [63]. In this context, identifying a specific genetic determinant that is responsible for a specific disease symptom under field or glasshouse conditions could be a real challenge [20].

Although serological or molecular markers were correlated with some CTV pathotypes [5], direct linkage of these markers to symptom development has not been established. Neverthe‐ less, the CP, p20 and p23, reported as suppressors of the plant silencing mechanism [18], could be candidates for symptom determinants since they could potentially disrupt the miRNA metabolism, thus possibly inducing disease. Indeed, several viral suppressors of RNAmediated gene silencing have been identified as pathogenicity determinants [66]. Actually, when p23 is ectopically expressed in transgenic limes or transgenic sour orange plants induces virus-like symptoms. However, the symptomatology pattern developed in these transgenic plants is different than those induced by natural virus infection. Additionally, the grade of symptom severity observed in these p23 transgenic plants is directly proportional to the p23 production level, and independent of the viral source or sequence of the p23 gene [67, 68]. Nevertheless, the symptom intensity in wild virus-infected limes or sour oranges is radically different between severe and mild isolates of virus. Yet, the different response in transgenic plants could be related to the fact that, in this case, the p23 protein is produced constitutively in most cells, while the expression of p23 is limited to phloem-associated cells in nature [20].

As previously described, a distinctive phenotype of some isolates of CTV is the ability to induce *Seedling yellows* in sour orange, lemon and grapefruit seedlings (Figure 6). To delimit the viral sequences associated with the SY syndrome, T36/T30 hybrids were generated by substituting severe sequences, located in the 3´ moiety of the T36-CTV9 infectious clone, for homologous asymptomatic sequences from the T30 genome. The T36/T30 hybrids were analyzed in *N. benthamiana* and citrus plants [20]. The SY determinant was mapped to the region encompass‐ ing the p23 gene and the 3' NTR (nt 18394-19296) (Figure 6) [20]. The 3'NTR has been used to generate transgenic plants resistant to CTV [69] and it has also been related to symptom development for other virus [70]. Likewise, the p23 is an obvious candidate for SY symptom determinant since it is one of the most highly expressed CTV proteins [54], a RNA-binding protein responsible for asymmetric replication [16, 17], and it is a viral suppressor of RNAmediated gene silencing mechanism [18]. Additionally, p23 ORF has been used to produce transgenic plants searching for resistance to CTV [71].

In order to map the stem pitting determinants, the effect on symptom development in *C. macrophylla* of deletions in p33, p18, and p13 ORFs were evaluated [21]. Although the T36 fulllength construct (*T36-CTV9*) causes only very mild SP symptoms in this host, certain deletion combinations (p33 and p18 and/or p13) greatly increased SP symptoms, while other combi‐ nations (p13 or p13 plus p18) resulted in reduced SP [21]. Remarkably, the stem-pitting phenotype seems to be induced as result of a balance between the expressions of different viral

**Figure 6.** Development of seedling yellows syndrome (SY) in CTV infected plants. Top panel: SY symptoms in (A) sour orange and (B) Duncan grapefruit seedlings compared to (C) a healthy sour orange plant. Bottom panel: SY symptoms in (1) T36/T30 hybrid [P23-3´NTR], (2) isolate T30 (3) healthy (4) T36 infectious clone CTV9 and (5) T36/T30 hybrid

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 15

There are different *Citrus* species and relatives that exhibit total or limited resistance to CTV infection. Pummelos, sour orange and the rootstock Swingle citrumelo display a differential degree of resistance depending on the CTV strain. However, some *Citrus* relatives, within subfamily *Aurantioideae,* like *P. trifoliata*, *Swinglea glutinosa* (Blanco) Merr., and *Severinia buxifolia* (Poir) Ten, as well as *P. trifoliata* intergenic hybrids like citranges (sweet orange × *P.*

genes.

**7.4. Host resistance to** *Citrus tristeza virus* **infection**

[HSP70h-P61] sour orange seedlings. From Albiach-Marti et al. [20].

*trifoliata*), remain resistant or immune to most of the CTV strains [4, 5].

The study of the devastating QD syndrome is especially important under de economical point of view. In this case, the extremely difficult task of reproducing this syndrome in glasshouse conditions hinders the study of the QD genetic determinants. However, since a strong correlation between SY and QD has been observed in the biological evaluation of a wide range of CTV isolates [5], it could be possible, but not yet confirmed, that determinant(s) for the decline disease map similarly to that of SY. Therefore, the CTV hybrids, used for evaluation of the SY genetics determinant [20], have been directly assessed in decline-susceptible grafted combinations of scion and rootstock in field conditions. In addition, since the hybrids are made by recombinant DNA technologies, these assays require special permits from the plant protection and environmental safety authorities [20].

**Figure 6.** Development of seedling yellows syndrome (SY) in CTV infected plants. Top panel: SY symptoms in (A) sour orange and (B) Duncan grapefruit seedlings compared to (C) a healthy sour orange plant. Bottom panel: SY symptoms in (1) T36/T30 hybrid [P23-3´NTR], (2) isolate T30 (3) healthy (4) T36 infectious clone CTV9 and (5) T36/T30 hybrid [HSP70h-P61] sour orange seedlings. From Albiach-Marti et al. [20].

In order to map the stem pitting determinants, the effect on symptom development in *C. macrophylla* of deletions in p33, p18, and p13 ORFs were evaluated [21]. Although the T36 fulllength construct (*T36-CTV9*) causes only very mild SP symptoms in this host, certain deletion combinations (p33 and p18 and/or p13) greatly increased SP symptoms, while other combi‐ nations (p13 or p13 plus p18) resulted in reduced SP [21]. Remarkably, the stem-pitting phenotype seems to be induced as result of a balance between the expressions of different viral genes.

#### **7.4. Host resistance to** *Citrus tristeza virus* **infection**

and about half of them without significant similarity with other known sequences [63]. In this context, identifying a specific genetic determinant that is responsible for a specific disease

Although serological or molecular markers were correlated with some CTV pathotypes [5], direct linkage of these markers to symptom development has not been established. Neverthe‐ less, the CP, p20 and p23, reported as suppressors of the plant silencing mechanism [18], could be candidates for symptom determinants since they could potentially disrupt the miRNA metabolism, thus possibly inducing disease. Indeed, several viral suppressors of RNAmediated gene silencing have been identified as pathogenicity determinants [66]. Actually, when p23 is ectopically expressed in transgenic limes or transgenic sour orange plants induces virus-like symptoms. However, the symptomatology pattern developed in these transgenic plants is different than those induced by natural virus infection. Additionally, the grade of symptom severity observed in these p23 transgenic plants is directly proportional to the p23 production level, and independent of the viral source or sequence of the p23 gene [67, 68]. Nevertheless, the symptom intensity in wild virus-infected limes or sour oranges is radically different between severe and mild isolates of virus. Yet, the different response in transgenic plants could be related to the fact that, in this case, the p23 protein is produced constitutively in most cells, while the expression of p23 is limited to phloem-associated cells in nature [20].

As previously described, a distinctive phenotype of some isolates of CTV is the ability to induce *Seedling yellows* in sour orange, lemon and grapefruit seedlings (Figure 6). To delimit the viral sequences associated with the SY syndrome, T36/T30 hybrids were generated by substituting severe sequences, located in the 3´ moiety of the T36-CTV9 infectious clone, for homologous asymptomatic sequences from the T30 genome. The T36/T30 hybrids were analyzed in *N. benthamiana* and citrus plants [20]. The SY determinant was mapped to the region encompass‐ ing the p23 gene and the 3' NTR (nt 18394-19296) (Figure 6) [20]. The 3'NTR has been used to generate transgenic plants resistant to CTV [69] and it has also been related to symptom development for other virus [70]. Likewise, the p23 is an obvious candidate for SY symptom determinant since it is one of the most highly expressed CTV proteins [54], a RNA-binding protein responsible for asymmetric replication [16, 17], and it is a viral suppressor of RNAmediated gene silencing mechanism [18]. Additionally, p23 ORF has been used to produce

The study of the devastating QD syndrome is especially important under de economical point of view. In this case, the extremely difficult task of reproducing this syndrome in glasshouse conditions hinders the study of the QD genetic determinants. However, since a strong correlation between SY and QD has been observed in the biological evaluation of a wide range of CTV isolates [5], it could be possible, but not yet confirmed, that determinant(s) for the decline disease map similarly to that of SY. Therefore, the CTV hybrids, used for evaluation of the SY genetics determinant [20], have been directly assessed in decline-susceptible grafted combinations of scion and rootstock in field conditions. In addition, since the hybrids are made by recombinant DNA technologies, these assays require special permits from the plant

transgenic plants searching for resistance to CTV [71].

protection and environmental safety authorities [20].

symptom under field or glasshouse conditions could be a real challenge [20].

14 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

There are different *Citrus* species and relatives that exhibit total or limited resistance to CTV infection. Pummelos, sour orange and the rootstock Swingle citrumelo display a differential degree of resistance depending on the CTV strain. However, some *Citrus* relatives, within subfamily *Aurantioideae,* like *P. trifoliata*, *Swinglea glutinosa* (Blanco) Merr., and *Severinia buxifolia* (Poir) Ten, as well as *P. trifoliata* intergenic hybrids like citranges (sweet orange × *P. trifoliata*), remain resistant or immune to most of the CTV strains [4, 5].

infection sites and they were usually single cells, indicating absence of cell to cell movement in both cases (Figure 7) [62]. However, the long-distance movement mechanism of CTV appears to be inefficient in some extend, since the majority of phloem-associated cells in the bark flaps have not been infected, even for *C. macrophylla* and Mexican lime susceptible hosts (Figure 7) [62]. In these experiments, accumulation of T36 seemed related to host susceptibility. Actually, the hypothesis points to plant silencing as a probable cause of this resistance mechanism [62]. However, inadequate interactions of the CTV host range determinants (p33, p18 and p13) [19] with the host factors, which allow viral movement, have not been discarded.

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 17

While CTV dispersal between new areas or countries occurs by graft propagation of virusinfected plant tissues, aphid transmission is responsible of local spread [1]. Viruliferous aphids of *Toxoptera citricida* (Kirkaldy) and *Aphis gossypii* (Glover) species are able to transmit CTV in a semipersistent manner [1] (Figure 1). However, *A. spiraecola* (Patch) and *T. aurantii* (Boyer de Fonscolombe) have also been reported as CTV vectors, although with less efficiency than *A. gossypii*. The aphid *T. citricida* is the most effective transmitting CTV and the most efficient and fast in the spatial and temporal viral spreading in citrus orchards*.* Moreover, when *T. citrici‐ da* appears in a new citrus area, the interaction between CTV and *T. citricida* seems to shift a specific mild or QD viral population to severe SP one [5]. This incidence suggests that *T. citricida* is more effective transmitting the minor virulent SP populations than the endemic mild or QD CTV genotypes. Citrus is the primary host of *T. citricida*, while *A. gossypii* populations build up in other crops. Probably *T. citricida* evolved with citrus and CTV and this could explain

The CTV genes or sequences related with aphid transmission are mostly unknown. However, usually for viral transmission, a *helper component* or the CTV virion has to interact with the mouthparts and the foregut of the aphids. Therefore, the protein components of the CTV particles (CP, CPm, HSP70h, p61) are candidates for aphid transmission determinants. In fact, CPm, which composes the particle tail structure of *Lettuce infectious yellows virus* (LIYV) (genus *Crinivirus*, family *Closteroviridae*), a close relative to CTV, is involved in viral transmission by *Bemisia tabaci* [75]. Similarly, the CTV CPm is suspected to affect aphid transmission [73, 74]. Comparison of CPm protein sequences from transmissible and non- transmissible CTV strains yield five mutations that appear to be conserved in transmissible CTV strains. These ones could affect aphid transmission efficiency by altering the conformation of the protein or masking motifs, which could be involved in the interaction between CPm and aphid stylet [76]. Although the special abilities of *T. citricida* are partially explained by its high efficiency in viral transmission [3], it seems that could be distinct interaction of this aphid with the coat proteins corresponding to different CTV genotypes. Additionally, the transmission mechanism of CTV

by *A. gossypii* may possibly be, to some extent, different to the one by *T. citricida*.

**8.** *Citrus tristeza virus* **genetic determinants related with aphid**

**transmission**

its high efficiency transmitting this virus [3].

**Figure 7.** Systemic infection of CTV in different citrus host. Top panel: localization of construct *CTV-BCN5-GFP*, derived from the recombinant virus *T36-CTV9, in* (A) leaf, (B) shoot and (C) roots of tolerant host *C. macrophylla*. Bottom panel: localization of *BCN5-GFP* in a bark flap of (D) Mexican lime, (E) *C. macrophylla*, (F) sweet orange, (G) sour orange and (H) Duncan grapefruit. Pictures were taken in a confocal microscope under UV light. Pictures from Folimonov et al. [9] and Folimonova et al. [62].

Resistance of plants to viruses results from blockage of a basic step in the virus life cycle. This blockage can result from the lack of a factor(s) in the plant that is necessary for virus multi‐ plication and movement (passive resistance) or activation of a defense mechanism (active resistance) [60]. One of the most effective methods of characterizing resistance mechanisms is to determine whether the resistance is expressed at the single-cell level. Albiach-Martí et al., [72] studied the nature of this CTV resistance mechanism and reported efficient multiplication of CTV in resistant *P. trifoliata* and its hybrids (Carrizo citrange, US119 and Swingle citrumelo) and *S. buxifolia* and *S. glutinosa* protoplasts (Figure 1). Thus, the resistance mechanism in these plant species affects a viral step subsequent to replication and assembly of viral particles, probably preventing CTV movement. Similar results were obtained in CTV inoculation experiments of resistant pummelo and sour orange protoplasts (Albiach-Martí, unpublished data). Likewise, the CTV systemic infection of Duncan grapefruit (a descent of pummelo) and sour orange plants was examined using a stable virus-based vector *CTV-BC5/GFP,* which was generated from the *T36-CTV9* recombinant virus (Figure 7) [9]. The susceptible host *C. macrophylla* and Mexican lime and the tolerant host sweet orange were used as controls [62]. CTV infection sites, after cell to cell movement, consisted of clusters of 3 to 12 cells in the susceptible species, while in Duncan grapefruit and sour orange there were fewer CTV infection sites and they were usually single cells, indicating absence of cell to cell movement in both cases (Figure 7) [62]. However, the long-distance movement mechanism of CTV appears to be inefficient in some extend, since the majority of phloem-associated cells in the bark flaps have not been infected, even for *C. macrophylla* and Mexican lime susceptible hosts (Figure 7) [62]. In these experiments, accumulation of T36 seemed related to host susceptibility. Actually, the hypothesis points to plant silencing as a probable cause of this resistance mechanism [62]. However, inadequate interactions of the CTV host range determinants (p33, p18 and p13) [19] with the host factors, which allow viral movement, have not been discarded.
