Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4-6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr-AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

Contribution of recombination and selection to molecular evolution of Citrus tristeza virus.

The genetic variation of Citrus tristeza virus (CTV) was analysed by comparing the predominant sequence variants in seven genomic regions (p33, p65, p61, p18, p13, p20 and p23) of 18 pathogenically distinct isolates from seven different countries. Analyses of the selective constraints acting on each codon suggest that most regions were under purifying selection. Phylogenetic analysis shows diverse patterns of molecular evolution for different genomic regions. A first clade composed of isolates that are genetically close to the reference mild isolates T385 or T30 was inferred from all genomic regions. A second clade, mostly comprising virulent isolates, was defined from regions p33, p65, p13 and p23. For regions p65, p61, p18, p13 and p23, a third clade that mostly included South American isolates could not be related to any reference genotype. Phylogenetic relationships among isolates did not reflect their geographical origin, suggesting significant gene flow between geographically distant areas. Incongruent phylogenetic trees for different genomic regions suggested recombination events, an extreme that was supported by several recombination-detecting methods. A phylogenetic network incorporating the effect of recombination showed an explosive radiation pattern for the evolution of some isolates and also grouped isolates by virulence. Taken together, the above results suggest that negative selection, gene flow, sequence recombination and virulence may be important factors driving CTV evolution.

Evidence of multiple recombination events between two RNA sequence variants within a Citrus tristeza virus isolate.

Analysis of sequence variants of a natural Citrus tristeza virus (CTV) isolate (SY568) revealed that its population was composed of three sequence types: (I) the most frequent type had > or =97.9% nucleotide identity with the sequence predominant in severe CTV isolates from different origins; (II) a second variant, genetically close to the major component of several mild isolates, had < or =85% identity with the first; and (III) several variants (less than 4%) resulted from homologous recombination at one or more sites between sequences I and II. Recombination sites had an AU-rich stretch of 8-89 nucleotides shared by both parental sequences, flanked by GC- and AU-rich regions upstream and downstream, respectively. This context has been suggested as a hot-spot for homologous recombination in other RNA viruses.

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.

Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus.

Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RP), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3'-coterminal and two 5'-coterminal subgenomic RNAs (sgRNAs). The 3'-coterminal sgRNAs contain the MP (3'MP sgRNA) and CP (3'CP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5' end. These sgRNAs start with a hexanucleotide which is also present at the 5' terminus of the gRNA. The 5'-coterminal sgRNAs have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5'RPMP sgRNA) and most ORF1 (5'RP sgRNA), respectively. Their 3' termini map 35 and 40 nt upstream of the transcription initiation of the 3'CP and 3'MP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3'-coterminal sgRNAs transcribed from the minus gRNA strand. The 5'-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis.