Dissecting aphid transmission determinants in Citrus Tristeza Virus using chimeric viruses and gene substitutions

Citrus Tristeza Virus (CTV) is the most economically significant viral pathogen of citrus worldwide, transmitted in a semipersistent manner by several aphid species, most efficiently by Aphis [Toxoptera] citricidus. Transmission efficiency varies substantially among CTV genotypes, but the viral determinants underlying these differences remain poorly understood. In this study, we investigated the roles of three viral genes, p33, p18, and p13, in aphid transmission using a genetically engineered CTV-T36 mutant (CTV-T36Δp33Δp18Δp13) as a backbone. This mutant is derived from the poorly transmissible T36 isolate and lacks three accessory genes implicated in host specificity and possibly vector interactions. To assess their contribution to transmissibility, we substituted the deleted genes with their counterparts from CTV-FS577, a highly transmissible isolate. The resulting chimeric virus exhibited a significant increase in transmission efficiency up to 50% compared to the original T36 background (∼0.6%). Sequence analysis revealed that among the three substituted genes, only p33 differs between T36 and FS577, with a single amino acid change (K174R). This residue is conserved in other highly transmissible isolates such as T68-1, supporting its potential role as a key determinant of transmission. Our findings highlight P33 as a critical viral protein in aphid-mediated transmission and suggest that efficient transmissibility requires coordinated function of multiple viral proteins, including P33, P61, and P65. This work provides new insights into the molecular basis of vector transmission and sets the stage for further studies on virus-vector interactions in CTV.