The work presented in this PhD memory is included in a project whose main objective is the development of an efficient viral vector based on Citrus Leaf Blotch Virus (CLBV) for expressing or silencing genes in citrus. We expect to use this vector as a genetic tool for: 1) rapid function evaluation of citrus genes or genes from other species that could be useful for the genetic improvement of citrus species, and 2) obtaining resistant or tolerant plants against citrus specific pathogens or diseases.  
To develop a viral vector based on CLBV genome it was necessary to have an infectious cDNA clone of the virus, and also efficient methods for inoculating CLBV in citrus plants. At the beginning of this work the only system available for citrus was an infectious clone from the Citrus tristeza virus (CTV). The only system allowing infectivity of this clone was inoculation of viral genomic RNA transcripts in Nicotiana benthamiana protoplasts, followed by amplification of the virions generated by successive serial passaging to new protoplasts and then mechanical virion inoculation to citrus plants. To reproduce the genetic system developed for CTV, we generated a cDNA infectious clone of the complete genome of CLBV under the T7 promoter of phage lambda, and we developed protocols for protoplast isolation from N. benthamiana, N. occidentalis and citron. CLBV replicated in protoplasts of these three species when transfected with purified virions, albeit replication level was low and CLBV could only be detected after 4-5 days post inoculation (dpi), which is the maximum time of protoplast survival in our experimental conditions. Viral multiplication in protoplasts inoculated with CLBV RNA transcripts was lower, and viral RNA could be detected only in a few transfections of N. benthamiana protoplasts. Finally, direct mechanical inoculation of citrus, N. benthamiana and N. occidentalis plants with viral RNA transcripts was unsuccessful, even if those transcripts were able to infect protoplasts. 
Before converting the CLBV infectious clone into an efficient viral vector, it was necessary a better understanding of the expression strategy of the viral genome and characterizing the sequences involved in promotion of subgenomic RNA (sgRNA) synthesis, to duplicate its promoter and express foreign genes or gene fragments by formation of a new sgRNA. In vivo characterization of the sequences involved in sgRNA promotion of plant viruses has been done by inoculating protoplasts with viral RNA transcripts. However, because CLBV accumulation level in protoplasts inoculated with viral transcripts was too low we couldn’t use this genetic system. Availability of an infectious cDNA clone of the complete CLBV genome under the CaMV 35S promoter (IC-CLBV) allowed us to develop a genetic system to characterize in vivo the sgRNA promoter of the CLBV coat protein gene (CP-sgRNA), by introducing the cDNA directly into the plant cells via Agrobacterium-mediated infiltration of N. benthamiana leaves. To map the CLBV CP-sgRNA promoter we generated several mutants from the IC-CLBV clone by nucleotide deletion and site-directed mutagenesis. Our results show that the CP-sgRNA promoter is located between nucleotides -67 and +50 from the transcription start site, and thus a 177 nucleotide segment or less would be enough to control the CP-sgRNA synthesis. Surprisingly, deletion of increasing fragments between the translation start site of the CP gene and the transcription start site of the CP-sgRNA induced higher sgRNA accumulation, suggesting that this sequence might modulate CP-sgRNA transcription. 
In the CLBV sequence, the GAAAAG hexanucleotide is present at the 5’ end of the gRNA and the two 3’ coterminal sgRNAs of the virus. To assess the importance of this hexanucleotide in the CP-sgRNA synthesis we generated mutants at different positions by deletion and site-directed mutagenesis. Mutation of the first and second nucleotides induced a drastic reduction in CP-sgRNA accumulation, suggesting that these nucleotides are important in the recognition by the CLBV RNA polymerase (RdRp) for CP-sgRNA transcription. On the other hand, elimination of an A from the hexanucleotide or mutations of G (+6) did not affect CP-sgRNA accumulation, suggesting that these nucleotides are not essential for RdRp recognition.  
Finally, to counteract plant defense mechanism based on posttranscriptional gene silencing, many viruses express proteins that inhibit this silencing at different levels. Viruses with strong silencing suppressors can be used as expression vectors but are not adequate as silencing inducing vectors (VIGS) because the suppressor might interfere with the plant silencing machinery (PTGS), thus impairing silencing of the gene of interest. Analysis of the silencing suppressor proteins of CLBV showed that only its movement protein was able to suppress intracellular PTGS, but not cell to cell- or long distance silencing. This protein showed a weak suppressor activity when compared to other well known silencing suppressor proteins. CLBV silencing suppressor protein acts interfering with host silencing machinery after siRNA (small interfering RNA) generation.