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

**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]

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

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results were obtained in CTV inoculation experiments of resistant pummelo and sour orange protoplasts (Albiach-Martí, unpublished data).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

and Folimonova et al. [62].

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 its high efficiency transmitting this virus [3].

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*.
