Luis Navarro

Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins

The population structure and genetic diversity of Citrus leaf blotch virus (CLBV) were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of two genomic regions located within the replicase (R) and the coat protein (C) genes. Analysis of 30 cDNA clones of each genomic region from two CLBV isolates showed that both isolates contained a predominant haplotype and others closely related. Analysis of 37 CLBV Spanish field isolates showed low genetic diversity (0.0041 and 0.0018 for genomic regions R and C, respectively). Comparison of 14 CLBV isolates from Spain, Japan, USA, France and Australia showed genetic diversities of 0.0318 (R) and 0.0209 (C), respectively. No correlation was found between genetic distance and geographical origin or host species of the isolates. The ratio between nonsynonymous and synonymous substitutions was the lowest found in a plant virus, indicating a strong negative selective pressure in both genomic regions.

RNAi-Mediated Protection Against Citrus Tristeza Virus in Transgenic Citrus Plants

RNA silencing is a sequence-specific mechanism of inhibition of gene expression evolutionarily conserved in most eukaryotes. RNA interference (RNAi), a technology based on the use of double-stranded RNA (dsRNA) to trigger RNA silencing, can be achieved in plants by genetic transformation with sense and antisense cDNAs derived from target viral sequences separated by an intron (intron-hairpin constructs). Upon transcription, the resulting hairpin RNA transcript usually acts as a strong inducer of RNA silencing. This strategy has been widely used to produce virus-resistant transgenic plants. Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of the most devastating viral diseases of citrus trees in the world. It only infects phloem-associated tissues of Citrus species and relatives within the family Rutaceae. CTV is one of the largest and most complex plant RNA viruses, with a single-stranded, plus-sense RNA genome of 19.3 kb, organized in 12 open reading frames (ORFs), potentially coding for at least 17 polypeptides, and two 5′ and 3′ unstranslated regions (UTRs). Replication and expression of the genomic RNA results in more than 30 different plus and minus RNA species, as well as their corresponding dsRNA forms. Concomitantly, citrus hosts have developed a strong antiviral response through RNA silencing, as inferred from the high level of virus-derived siRNAs observed in infected tissues. As a counterdefense, CTV encodes at least three silencing suppressor proteins acting intracellularly and/or intercellularly to overcome antiviral defense. Under these circumstances, searching for RNAi-induced resistance against CTV in transgenic citrus plants becomes a real challenge. We have used intron-hairpin constructs targeting several viral regions, with our present interest focusing on one or the three CTV genes encoding silencing suppressors, or on conserved domains important for viral replication and encapsidation.