Supriya Chakraborty

Tomato cultivar tolerant to Tomato leaf curl New Delhi virus infection induces virus‐specific short interfering RNA accumulation and defence‐associated host gene expression

Tomato leaf curl New Delhi virus (ToLCNDV) infection causes significant yield loss in tomato. The availability of a conventional tolerance source against this virus is limited in tomato. To understand the molecular mechanism of virus tolerance in tomato, the abundance of viral genomic replicative intermediate molecules and virus-directed short interfering RNAs (siRNAs) by the host plant in a naturally tolerant cultivar H-88-78-1 and a susceptible cultivar Punjab Chhuhara at different time points after agroinfection was studied. We report that less abundance of viral replicative intermediate in the tolerant cultivar may have a correlation with a relatively higher accumulation of virus-specific siRNAs. To study defence-related host gene expression in response to ToLCNDV infection, the suppression subtractive hybridization technique was used. A library was prepared from tolerant cultivar H-88-78-1 between ToLCNDV-inoculated and Agrobacterium mock-inoculated plants of this cultivar at 21 days post-inoculation (dpi). A total of 106 nonredundant transcripts was identified and classified into 12 different categories according to their putative functions. By reverse Northern analysis and quantitative real-time polymerase chain reaction (qRT-PCR), we identified the differential expression pattern of 106 transcripts, 34 of which were up-regulated (>2.5-fold induction). Of these, eight transcripts showed more than four fold induction. qRT-PCR analysis was carried out to obtain comparative expression profiling of these eight transcripts between Punjab Chhuhara and H-88-78-1 on ToLCNDV infection. The expression patterns of these transcripts showed a significant increase in differential expression in the tolerant cultivar, mostly at 14 and 21 dpi, in comparison with that in the susceptible cultivar, as analysed by qRT-PCR. The probable direct and indirect relationship of siRNA accumulation and up-regulated transcripts with the ToLCNDV tolerance mechanism is discussed.

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

BackgroundEmerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.ResultsWe demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.ConclusionThis is the first report and experimental demonstration of Kochs postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.

A geminivirus betasatellite damages the structural and functional integrity of chloroplasts leading to symptom formation and inhibition of photosynthesis

Highlight A satellite DNA-encoded protein (βC1) is localized in the chloroplast. The intercellular events associated with βC1-induced photosynthetic inhibition and vein clearing symptom formation are discussed.

Molecular characterization of a new species of Begomovirus and betasatellite causing leaf curl disease of tomato in India.

A new tomato-infecting begomovirus and cognate betasatellite were characterized from the gangetic plain of northern India. Genome organization of this virus was found to be similar to those of other old world begomoviruses. The DNA-A molecule (2752nt) shared maximum (85.8%) identity with Tomato leaf curl Laos virus-[Laos] (ToLCLV-[LA]; AF195782) from Laos and betasatellite molecule (1349nt) shared maximum (75.8%) identity with Tomato leaf curl Joydebpur betasatellite (ToLCJoB-[BD:Gaz:05]; AJ966244) from Bangladesh. Interestingly, both these molecules showed less identity with known tomato-infecting begomoviruses and their satellites from India. The recombination detection program (RDP) revealed that these molecules are not an outcome of direct exchange of sequences between existing begomovirus species. According to International Committee on Taxonomy of viruses (ICTV) species/strains demarcations norms for viruses belonging to the family Geminiviridae, this is a new Begomovirus species and we named this virus as Tomato leaf curl Patna virus (ToLCPaV) and new beta species as Tomato leaf curl Patna betasatellite (ToLCPaB). Partial tandem repeats of ToLCPaV and ToLCPaB could induce typical leaf curl symptom on tomato (Solanum lycopersicum) and Nicotiana benthamiana. Although, DNA-A could alone infect tomato typical to a monopartite Begomovirus, co-inoculation of DNA-A and DNA-beta resulted more stunting and severe symptoms. Interestingly, association of ToLCPaB did not assist in increased ToLCPaV accumulation in systemic leaves. ToLCPaV neither transreplicate DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) nor of Tomato leaf curl Gujarat virus (ToLCGV), presumably due to difference in rep-binding sequences. However, ToLCPaB formed viable pseudorecombinant with mono-bipartite ToLCGV DNA-A infecting both N. benthamiana and tomato but could not cause systemic infection on natural host tomato when co-inoculated with ToLCNDV DNA-A, which is a bipartite Begomovirus.

A novel recombinant tomato-infecting begomovirus capable of transcomplementing heterologous DNA-B components

The genome of a tomato-infecting begomovirus from Ranchi, India, was cloned, sequenced and analysed. The viral genome shared 88.3% sequence identity with an isolate belonging to the species Tobacco curly shoot virus (TbCSV), and this virus should therefore be considered a member of a new species, tentatively named Tomato leaf curl Ranchi virus (ToLCRnV). The DNA-β molecule, which had 74.5% sequence identity with tomato leaf curl Bangladesh betasatellite (ToLCBDB), is named tomato leaf curl Ranchi betasatellite (ToLCRnB). Phylogenetic analysis revealed that ToLCRnV is related to tomato leaf curl Bangladesh virus (ToLCBDV), tobacco curly shoot virus (TbCSV) and tomato leaf curl Gujarat virus (ToLCGV). An infectivity study with ToLCRnV established the monopartite nature of the viral genome, whereas inoculation with ToLCRnB resulted in increased symptom severity. ToLCRnV could transreplicate DNA-B of tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl New Delhi virus (ToLCNDV), both in N. benthamiana and tomato, although DNA-B accumulation of was less than with the wild-type combinations. ToLCRnB could be efficiently replicated by DNA-A of both ToLCNDV and ToLCGV. A leaf disk assay suggests that DNA-A could transreplicate the homologous DNA-B and DNA-β more efficiently than the heterologous one.

Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India

Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report on a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60 × 10- 3 substitutions site- 1 year- 1) and the βC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57 × 10- 4 substitution site- 1 year- 1; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22 × 10- 4 substitution site- 1 year- 1]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.

Biological and molecular characterization of a begomovirus associated with yellow mosaic vein mosaic disease of pumpkin from Northern India

The biological and molecular properties of Squash leaf curl China virus from Varanasi, India (SLCCNV-IN[IN:Var:Pum]) were characterized. SLCCNV-IN[IN:Var:Pum] could be transmitted by grafting and through whitefly transmission. The complete DNA-A and DNA-B components were amplified through PCR using specific DNA-A and DNA-B primers. The DNA-A of the isolate was comprised of 2,738 nucleotides, encoding typical six open reading frames, and DNA-B of 2,704 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region have the highest sequence identity (97.6 and 97.4%, respectively) with the SLCCNV-IN[IN:Luc:Pum]; (DQ026296). This data suggested that the isolate is a same begomovirus species for which the name Squash leaf curl China virus-India[India:Varanasi:Pumpkin] is proposed. DNA-B showed maximum sequence identity (89.2%) with SLCCNV-IN[IN:Coi:Pum] (AY184488). Phylogenetic analysis of the present isolate showed close relationship to other cucurbit-infecting geminiviruses. This is the first evidence of occurrence of the bipartite Squash leaf curl China virus associated with severe yellow mosaic disease of pumpkin in northern India.

Differential response of diverse solanaceous hosts to tomato leaf curl New Delhi virus infection indicates coordinated action of NBS-LRR and RNAi-mediated host defense

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) that infects a wide range of plants. ToLCNDV has emerged as an important pathogen and a serious threat to tomato production in India. A comparative and molecular analysis of ToLCNDV pathogenesis was performed on diverse solanaceous hosts (Capsicum annuum, Nicotiana benthamiana,N. tabacum, and Solanum lycopersicum). N. benthamiana was found to be the most susceptible host, whereas C. annuum showed resistance against an isolate of ToLCNDV collected in New Delhi from tomato (GenBank accession no. U15015 and U15017). S. lycopersicum and N. tabacum developed conspicuous symptoms and allowed virus to accumulate to significantly high titers. The viral DNA level was concurrent with symptom severity. ToLCNDV-specific siRNA levels were directly proportional to the amount of viral DNA. To investigate the basis for the differences in response of these hosts to ToLCNDV, a comparative expression analysis of selected defense-related genes was carried out. The results indicated differences in expression levels of genes involved in the posttranscriptional gene silencing machinery (RDR6, AGO1 and SGS3) as well as basal host defense responses (nucleotide-binding site and leucine-rich repeat [NBS-LRR] proteins and lipid transfer protein [LTP]). Among these, expression of NBS-LRR genes was found to be significantly higher in C. annuum following ToLCNDV infection. Our analyses suggest that the expression of host defense responses determines the level of ToLCNDV accumulation and degree of symptom development.

Occurrence and analysis of imperfect microsatellites in diverse potyvirus genomes

Simple sequence repeats (SSRs) or microsatellites are known to exhibit ubiquitous across all kingdoms of life including viruses. However, imperfections in simple sequence repeats have been analyzed in genomes of human, Escherichia coli and Human Immunodeficiency virus. The assessment of compound microsatellites in plant viral genomes is yet to be studied. Potyviruses severely affect crop plant growth and reduce economic yield in diverse cropping systems worldwide. Hence, we analyze the nature and distribution of compound microsatellites present in complete genome of 45 potyvirus species. The results indicate that compound microsatellites accounted for about 0% to 15.15% of all microsatellites and have low complexity as compared to that of prokaryotic genomes. Overall, 14% of compound microsatellites were of similar motifs and such motif duplications were observed for CA, TA and AG repeats. Among all 45 potyvirus genomes analyzed, SSR couple (AG)-x-(AC) was found to be the most abundant one. Hence it is apparent that in contrast to eukaryotes, majority of compound microsatellites in potyviruses were composed of variant motifs. We also highlight the relative frequency of different classes of compound microsatellites as well as their patterns of distribution and correlate with biology of potyviruses. Further characterization of such variation is important for elucidating the origin, mutational processes, and structure of these widely used, but incompletely understood sequences.

Tomato Leaf Curl Viruses from India

Leaf curl disease of tomato (ToLCD) caused by tomato leaf curl viruses (ToLCVs) is one of the major constraints for successful cultivation of this crop throughout India. Intensive agricultural practices necessitated by the ever-increasing demands of rapidly growing population and the introduction of new genotypes and cropping pattern have further aggravated the situation. Whitefly (Bemisia tabaci Genn.) can successfully transmit the causal virus under natural conditions. Several crop as well as weed species are known to harbor ToLCVs in India. Maximum temperature and rainfall play an important role for spread of the disease in southern India, while minimum temperature and minimum relative humidity influence the whitefly population in the north. Five distinct species of the genus Begomovirus (Tomato leaf curl Bangalore virus, Tomato leaf curl Karnataka virus, Tomato leaf curl Gujarat virus, Tomato leaf curl Pune virus, and Tomato leaf curl New Delhi virus) are known to be associated with ToLCD in India. Satellite DNA-βs associated with ToLCVs are highly diverse and share a much lower degree of nucleotide identity among themselves. Mixed begomovirus infections, a prerequisite for recombination, are also observed which lead to synergistic interaction and virulent pseudo-recombination. Extensive research on conventional breeding has led to development of resistant genotypes from which resistant gene has also been mapped.