CITRUS LEAF BLOTCH VIRUS: A CITRUS PATHOGEN TRANSFORMED IN A GENETIC TOOL

Citrus leaf blotch virus (CLBV), the type member of genus Citrivirus, family Flexiviridae, was initially purified from a Nagami kumquat from Corsica that induced chlorotic blotching in Dweet tangor, stem pitting in Etrog citron, vein clearing in Pineapple sweet orange and other cultivars, and bud union crease when propagated on Troyer citrange. This pathogen was also detected in commercial orchards of different geographic areas affecting different citrus varieties grafted on trifoliate rootstocks that showed bud union crease. CLBV has a single-stranded, positive sense, genomic RNA (gRNA) of 9 Kb organized in three ORFs encoding a polyprotein involved in replication, a movement protein (MP) and the coat protein (CP). Availability of the gRNA sequence allowed molecular diagnostics by hybridization, RT-PCR, and quantitative real time RT-PCR, and the preparation of a full-genome cDNA clone that was inserted in a binary vector. Agro-inoculation of this vector in leaves of citrus and Nicotiana benthamiana resulted in systemic infection and enabled demonstrating that CLBV actually induces chlorotic blotching in Dweet tangor and stem pitting in Etrog citron, but not vein clearing in Pineapple sweet orange or bud union crease on trifoliate rootstocks, suggesting that the two latter symptoms are likely caused by a different pathogen or by an interaction between CLBV and other biotic factors. Recently we have modified the sequence of the infectious clone adding a unique restriction site after the stop codon of the CP gene, or in the intergenic region between the MP and the CP genes, in order to obtain a viral vector capable of silencing citrus genes. We have also duplicated the promoter sequence of the CP gene at these points to express foreign genes in citrus plants. GFP expression from these viral vectors enabled the study of CLBV movement in N. benthamiana plants. The potential use of the infectious clone as a genetic tool for citrus will be discussed.