Hormonal and metabolic responses of Mexican lime plants to CTV infection.

Plant viral infections alter gene expression and metabolism in infected host. To study the molecular responses of Mexican lime to CTV infection, an analysis of plant metabolome in response to infection with severe (T318) or mild (T385) isolates of CTV was performed. Healthy plants and those infected with any of the two virus strains showed different metabolite profiles, at different stages of new sprout development. Proline content increased in plants infected with CTV, proportionally to the virulence of the virus strain. Abscisic acid content decreased after virus infection whereas jasmonic and salicylic acid levels increased. CTV infection had an impact on plant secondary metabolism, by stimulating the synthesis of different metabolites such as l-methylhistidine, phenylpropanoid derivatives. These metabolites are common responses of different organisms, including higher mammals, to viral diseases, and its presence in this system points to the existence of universal responses to virus infection among different kingdoms.

Codon Usage Bias Analysis of Citrus tristeza Virus: Higher Codon Adaptation to Citrus reticulata Host.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host⁻virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3'-terminal region of the genomic RNA and affects the host small RNA profile.

To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21-22-nt, with a biased distribution of their 5' nucleotide and with those of (+) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20 kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 3'-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 3' co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation.

Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

Sequencing and computational analysis of complete genome sequences of Citrus yellow mosaic badna virus from acid lime and pummelo.

Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus, is the causative agent of Citrus mosaic disease in India. Although the virus has been detected in several citrus species, only two full-length genomes, one each from Sweet orange and Rangpur lime, are available in publicly accessible databases. In order to obtain a better understanding of the genetic variability of the virus in other citrus mosaic-affected citrus species, we performed the cloning and sequence analysis of complete genomes of CMBV from two additional citrus species, Acid lime and Pummelo. We show that CMBV genomes from the two hosts share high homology with previously reported CMBV sequences and hence conclude that the new isolates represent variants of the virus present in these species. Based on in silico sequence analysis, we predict the possible function of the protein encoded by one of the five ORFs.

Grouping and comparison of Indian citrus tristeza virus isolates based on coat protein gene sequences and restriction analysis patterns.

Citrus tristeza virus (CTV) is an aphid-transmitted closterovirus, which causes one of the most important citrus diseases worldwide. Isolates of CTV differ widely in their biological properties. CTV-infected samples were collected from four locations in India: Bangalore (CTV-B), Delhi (CTV-D), Nagpur (CTV-N), and Pune (CTV-P), and were maintained by grafting into Kagzi lime ( Citrus aurantifolia (Christm. Swing.). All isolates produced typical vein clearing and flecking symptoms 6-8 weeks after grafting. In addition, CTV-B and CTV-P isolates produced stem-pitting symptoms after 8-10 months. The CTV coat protein gene (CPG) was amplified by RT-PCR using CPG specific primers, yielding an amplicon of 672 bp for all the isolates. Sequence analysis of the CPG amplicon of all the four Indian isolates showed 93-94% nucleotide sequence homology to the Californian CTV severe stem pitting isolate SY568 and 92-93% homology to the Japanese seedling yellows isolate NUagA and Israeli VT p346 isolates. In phylogenetic tree analysis, Indian CTV isolates appeared far different from other isolates as they formed a separate branch. Comparison among the Indian isolates was carried out by restriction analysis and restriction fragment length polymorphism (RFLP). Specific primers to various genome segments of well-characterized CTV isolates were used to further classify the Indian CTV isolates.