Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.
ABSTRACT: Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato.
Project description: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.
Project description:Tomato leaf curl New Delhi virus (ToLCNDV) is an exceptional Old World bipartite begomovirus. On the Indian subcontinent, a region in which monopartite DNA satellite-associated begomoviruses with mostly narrow geographical ranges predominate, it is widespread, with a geographical range also including the Far East, Middle East, North Africa and Europe. The success of ToLCNDV probably derives from its broad host range and highly flexible genomic configuration: its DNA-A component is capable of productively interacting with, and trans-replicating, diverse DNA-B components and betasatellites. An understanding of the capacity of ToLCNDV to infect a variety of hosts and spread across a broad and ecologically variable geographical range could illuminate the potential economic threats associated with similar begomoviral invasions. Towards this end, we used available ToLCNDV sequences to reconstruct the history of ToLCNDV spread. TAXONOMY:Family Geminiviridae, Genus Begomovirus. ToLCNDV is a bipartite begomovirus. Following the revised begomovirus taxonomic criteria of 91% and 94% nucleotide identity for species and strain demarcation, respectively, ToLCNDV is a distinct species with two strains: ToLCNDV and ToLCNDV-Spain. HOST RANGE:The primary cultivated host of ToLCNDV is tomato (Solanum lycopersicum), but the virus is also known to infect 43 other plant species from a range of families, including Cucurbitaceae, Euphorbiaceae, Solanaceae, Malvaceae and Fabaceae. DISEASE SYMPTOMS:Typical symptoms of ToLCNDV infection in its various hosts include leaf curling, vein thickening, puckering, purpling/darkening of leaf margins, leaf area reduction, internode shortening and severe stunting.
Project description:The tomato leaf curl New Delhi virus (ToLCNDV) (genus Begomovirus, family Geminiviridae) represents an important constraint to tomato production, as it causes the most predominant and economically important disease affecting tomato in the Indian sub-continent. However, in recent years, ToLCNDV has been fast extending its host range and spreading to new geographical regions, including the Middle East and the western Mediterranean Basin. Extensive research on the genome structure, protein functions, molecular biology, and plant-virus interactions of ToLCNDV has been conducted in the last decade. Special emphasis has been given to gene silencing suppression ability in order to counteract host plant defense responses. The importance of the interaction with DNA alphasatellites and betasatellites in the biology of the virus has been demonstrated. ToLCNDV genetic variability has been analyzed, providing new insights into the taxonomy, host adaptation, and evolution of this virus. Recombination and pseudorecombination have been shown as motors of diversification and adaptive evolution. Important progress has also been made in control strategies to reduce disease damage. This review highlights these various achievements in the context of the previous knowledge of begomoviruses and their interactions with plants.
Project description:BACKGROUND: Begomoviruses have emerged as serious problem for vegetable and fiber crops in the recent past, frequently in tropical and subtropical region of the world. The association of begomovirus with eggplant yellow mosaic disease is hitherto unknown apart from one report from Thailand. A survey in Nagpur, Central India, in 2009-2010 showed severe incidence of eggplant yellow mosaic disease. Here, we have identified and characterized a begomovirus responsible for the newly emerging yellow mosaic disease of eggplant in India. RESULTS: The complete DNA-A and DNA-B genomic components of the causative virus were cloned and sequenced. Nucleotide sequence analysis of DNA-A showed that it shared highest 97.6% identity with Tomato leaf curl New Delhi virus-India[India:Udaipur:Okra:2007] and lowest 87.9% identity with Tomato leaf curl New Delhi virus-India[India:NewDelhi:Papaya:2005], while DNA-B showed highest 94.1% identity with ToLCNDV-IN[IN:UD:Ok:07] and lowest 76.2% identity with ToLCNDV-India[India:Lucknow]. Thus, it appears that this begomovirus is a variant of ubiquitous ToLCNDV and hence, we suggest the name ToLCNDV-India[India:Nagpur:Eggplant:2009] for this variant. The pathogenicity of ToLCNDV-IN[IN:Nag:Egg:09] isolate was confirmed by agroinfiltraion and dimeric clones of DNA-A and DNA-B induced characteristic yellow mosaic symptoms in eggplants and leaf curling in tomato plants. CONCLUSION: This is the first report of a ToLCNDV variant moving to a new agriculturally important host, eggplant and causing yellow mosaic disease. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with eggplant yellow mosaic disease.
Project description:During 2012-2014, mosaic disease on chayote in the farmers field of Kodaikanal region (high altitude zone) of Tamil Nadu was observed. The disease was characterized with severe mosaic, cupping and enation on leaves with reduced fruit size. Disease was found to causes an yield loss of more than 60% with the maximum disease incidence of 100% for the past 5 years consecutively. Preliminary serological and molecular screening indicated the association of begomovirus with the disease. Complete nucleotide sequence and phylogenetic analysis of DNA A revealed the identity of the virus as tomato leaf curl New Delhi virus (ToLCNDV). In recombination analysis study, the major parent was identified as ToLCNDV from Pakistan infecting tomato. Thus the present finding confirms expansion of new geographical region and host for ToLCNDV causing mosaic disease on chayote from Tamil Nadu. To our knowledge this is the first confirmed report for the occurrence of ToLCNDV on chayote in southern India.
Project description:An evaluation of 70 accessions of ash gourd germplasm grown at National Bureau of Plant Genetic Resources, New Delhi, India during Kharif season (2010) showed natural occurrence of a yellow stunt disease in three accessions (IC554690, IC036330 and Pusa Ujjwal). A set of begomovirus specific primers used in PCR gave expected amplicon from all the symptomatic plants; however no betasatellite was detected. Complete genome of the begomovirus (DNA-A and DNA-B), amplified through rolling circle amplification, was cloned and sequenced. The begomovirus under study shared high sequence identities to different isolates of Tomato leaf curl New Delhi virus (ToLCNDV) and clustered with them. Among those isolates, the DNA-A and DNA-B of the present begomovirus isolate showed highest 99.6 and 96.8 % sequence identities, respectively with an isolate reported on pumpkin from India (DNA-A: AM286433, DNA-B: AM286435). Based on the sequence analysis, the begomovirus obtained from ash gourd was considered as an isolate of ToLCNDV. Thus, the present findings constitute the first report of occurrence of a new yellow stunt disease in ash gourd from India and demonstrated the association of ToLCNDV with the symptomatic samples. Occurrence of ToLCNDV in ash gourd germplasm not only adds up a new cucurbitaceous host of this virus but also raises the concern about the perpetuation of this virus in absence of its main host tomato and thus has an epidemiological relevance for understanding the rapid spread of this virus in tomato and other hosts in Indian sub-continent.
Project description:During 2006, pumpkin leaf curl-a new disease was observed in the experimental field at Indian Agricultural Research Institute. The disease was characterized by upward leaf curl with chlorotic patches and stunting of plant. Polymerase chain reaction (PCR) with coat protein specific primers to Tomato leaf curl New Delhi virus (ToLCNDV) indicated association of a begomovirus with the disease. The sequence comparison and phylogenetic analysis of the complete DNA genome further revealed the identity of the virus as ToLCNDV. The study provides evidence that ToLCNDV is associated with the leaf curl of pumpkin (Cucurbita moschata) in northern India.
Project description:BACKGROUND: Tomato leaf curl virus (ToLCV), a constituent of the genus Begomovirus, infects tomato and other plants with a hallmark disease symptom of upward leaf curling. Since microRNAs (miRs) are known to control plants developmental processes, we evaluated the roles of miRNAs in Tomato leaf curl New Delhi virus (ToLCNDV) induced leaf curling. RESULTS: Microarray analyses of miRNAs, isolated from the leaves of both healthy and ToLCNDV agroinfected tomato cv Pusa Ruby, revealed that ToLCNDV infection significantly deregulated various miRNAs representing ~13 different conserved families (e.g., miR319, miR172, etc.). The precursors of these miRNAs showed similar deregulated patterns, indicating that the transcription regulation of respective miRNA genes was perhaps the cause of deregulation. The expression levels of the miRNA-targeted genes were antagonistic with respect to the amount of corresponding miRNA. Such deregulation was tissue-specific in nature as no analogous misexpression was found in flowers. The accumulation of miR159/319 and miR172 was observed to increase with the days post inoculation (dpi) of ToLCNDV agroinfection in tomato cv Pusa Ruby. Similarly, these miRs were also induced in ToLCNDV agroinfected tomato cv JK Asha and chilli plants, both exhibiting leaf curl symptoms. Our results indicate that miR159/319 and miR172 might be associated with leaf curl symptoms. This report raises the possibility of using miRNA(s) as potential signature molecules for ToLCNDV infection. CONCLUSIONS: The expression of several host miRNAs is affected in response to viral infection. The levels of the corresponding pre-miRs and the predicted targets were also deregulated. This change in miRNA expression levels was specific to leaf tissues and observed to be associated with disease progression. Thus, certain host miRs are likely indicator of viral infection and could be potentially employed to develop viral resistance strategies.
Project description:RNA interference (RNAi), a conserved RNA-mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter-strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down-regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans-acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single-step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro-infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro-infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA-generating constructs, and T1 plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.
Project description:This is the first report of a begomovirus infecting luffa in Indonesia. The genome of this virus shares a close identity with that of Tomato leaf curl New Delhi virus (ToLCNDV). There is a 36-nucleotide duplicated sequence in the DNA-B component, suggesting the occurrence of an intraviral recombination.