Project description:Capsicum annuum, a valuable spice and vegetable crop belonging to the Solanaceae family, is extensively grown across the Indian subcontinent. Chilli production is restricted by a begomoviral infection named as chilli leaf curl disease (ChiLCD) mainly in tropical and subtropical regions which leads to considerable economic losses, thus affecting chilli cultivation. Here, we studied the genetic diversity with structural evaluation of chilli leaf curl disease and satellite molecules infecting Chilli in India. We retrieved 121 reference sequences of ChiLCD including DNA-A, DNA-B, beta-satellite and alpha-satellites from GenBank reported from India. The population diversity and genetic variation were estimated through various parameters which decipher the four major groups of phylogenetic divergence for DNA-A and five groups of beta-satellite showing percentage similarity with isolates within and across India. Further, transitional and transversional bias for ORFs were observed highest in C4 and REn genes, respectively, and for DNA-A and DNA-B, these values were 1.07 and 1.22, respectively. The recombination breakpoints for DNA-A were estimated 49 majorly in V1, C1,C2 and C4 genome region and highest 22 breakpoints were determined for Rep (AC1) of ORFs, similarly 9 events for beta-satellite were found less around βC1ORF. Moreover, the evolution and genetic variability were also contributed through parameters such as nucleotide substitution which were found within the range of RNA viruses for DNA-A, DNA-B, for all 6 ORFs (relaxed clock) and beta-satellite. Additionally, total numbers of mutations (η) for DNA-A, DNA-B, alpha-satellites and beta-satellites were 2505, 419, 807 and 1288 detected, respectively, while it was found 987 highest for Rep gene among all ORFs. Further, neutrality tests determine the dominant nature of population expansion and purifying selection for all the genes of begomovirus associated with ChiLCD and satellite molecules supporting conserved nature of gene. The combined Tajima's D and Fu and Li'S D* negative values in tests indicated that population are under purified selection and an excess of low-frequency polymorphism. Our analysis indicates the potential contribution of genetic mutations and recombination of ChiLCD which leads to rapid adaptation and evolution of begomovirus and its satellite molecules accelerating its host range and diversity within and across the Indian subcontinent.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03139-w.
Project description:Chilli (Capsicum annum L.) is well known as 'wonder spice'. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.
Project description:BACKGROUND:In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. METHODS:Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. RESULTS:The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. CONCLUSIONS:The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.
Project description:Pepper leaf curl virus (PepLCV) is a serious threat to pepper (Capsicum spp.) production worldwide. Molecular mechanism underlying pepper plants response to PepLCV infection is key to develop PepLCV resistant varieties. In this study, we generated transcriptome profiles of PepLCV resistant genotype (BS-35) and susceptible genotype (IVPBC-535) after artificial viral inoculation using microarray technology and detail experimental procedures and analyses are described. A total of 319 genes differentially expressed between resistant and susceptible genotypes were identified, out of that 234 unique genes were found to be up-regulated > 2-fold in resistant line BS-35 when compared to susceptible, IVPBC-535. The data set we generated has been analyzed to identify genes that are involved in the regulation of resistance against PepLCV. The raw data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE41131.
Project description:Chilli leaf curl virus (ChiLCV), (Genus Begomovirus, family Geminiviridae) and associated satellites pose a serious threat to chilli production, worldwide. This study highlights the factors accountable for genetic diversity, recombination, and evolution of ChiLCV, and associated chilli leaf curl alphasatellite (ChiLCA) and chilli leaf curl betasatellite (ChiLCB). Phylogenetic analysis of complete genome (DNA-A) sequences of 132 ChiLCV isolates from five countries downloaded from NCBI database clustered into three major clades and showed high population diversity. The dN/dS ratio and Tajima D value of all viral DNA-A and associated betasatellite showed selective control on evolutionary relationships. Negative values of neutrality tests indicated purified selection and an excess of low-frequency polymorphism. Nucleotide diversity (π) for C4 and Rep genes was higher than other genes of ChiLCV with an average value of π = 18.37 × 10-2 and π = 17.52 × 10-2 respectively. A high number of mutations were detected in TrAP and Rep genes, while ChiLCB has a greater number of mutations than ChiLCA. In addition, significant recombination breakpoints were detected in all regions of ChiLCV genome, ChiLCB and, ChiLCA. Our findings indicate that ChiLCV has the potential for rapid evolution and adaptation to a range of geographic conditions and could be adopted to infect a wide range of crops, including diverse chilli cultivars.
Project description:Geminiviruses are single-stranded DNA viruses that can cause significant losses in economically important crops. In recent years, the role of different kinases in geminivirus pathogenesis has been emphasized. Although geminiviruses use several host kinases, the role of phosphatidylinositol 4-kinase (PI4K) remains obscure. We isolated and characterized phosphatidylinositol 4-kinase type II from Nicotiana benthamiana (NbPI4KII) which interacts with the replication initiator protein (Rep) of a geminivirus, chilli leaf curl virus (ChiLCV). NbPI4KII-mGFP was localized into cytoplasm, nucleus or both. NbPI4KII-mGFP was also found to be associated with the cytoplasmic endomembrane systems in the presence of ChiLCV. Furthermore, we demonstrated that Rep protein directly interacts with NbPI4KII protein and influenced nuclear occurrence of NbPI4KII. The results obtained in the present study revealed that NbPI4KII is a functional protein kinase lacking lipid kinase activity. Downregulation of NbPI4KII expression negatively affects ChiLCV pathogenesis in N. benthamiana. In summary, NbPI4KII is a susceptible factor, which is required by ChiLCV for pathogenesis.
Project description:A resistant source (S-343) having monogenic dominant resistance to chilli leaf curl virus disease (ChiLCVD) has been identified at Punjab Agricultural University (PAU), Ludhiana. The F2 mapping population of 204 plants was derived from the cross MS-341 (susceptible) × S-343 (resistant) to identify the linked marker with the disease-resistant gene. Out of the 685 single-sequence repeats (SSRs) used, only 160 primers showed parental polymorphism. These 160 polymorphic primers were used for bulk segregant analysis and only eight SSR primers were able to differentiate the resistant and susceptible bulks. The linkage analysis revealed that the two markers CA 516044 and PAU-LC-343-1 were found linked with the disease-resistant gene covering a total distance of 15.7 centimorgan (cM). The two primers CA 516044 and PAU-LC-343-1 were found located on chromosome 6 of the pepper genome at a genetic distance of 6.8 cM and 8.9 cM, respectively, from the resistant gene. The validation of linked markers was performed using 26 resistant and susceptible genotypes developed at PAU, Ludhiana by former researchers. The validation of the primers revealed that there was a correlation between phenotypic and genotypic data of the used genotypes, and these markers can be used for the marker-assisted breeding procedures for transferring ChiLCVD resistance until the gene-based markers will be developed. The markers described in this study are the first-ever molecular markers identified as linked to the ChiLCVD-resistant gene.
Project description:Cotton leaf curl disease (CLCuD) is one of the major limiting factors affecting cotton production in Pakistan for the last three decades. The disease is caused by begomoviruses of the family Geminiviridae. RNA interference (RNAi) is a promising tool that has been proved effective against several pathogens. Using RNAi, different genomic regions of geminiviruses have been targeted to attain sustainable resistance. However, the silencing of the transgene upon virus infection is a limiting factor. Here, we have developed for the first time an amplicon-based RNAi construct to target βC1 gene of betasatellite associated with cotton leaf curl begomoviruses. In addition to producing short interfering (si) RNAs, Rep-based activation or looping out of the construct induced upon virus infection produces multiple copies of transgene that results in accumulation of defective molecules of betasatellite. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Transgenic Nicotiana benthamiana plants having RCβ (RNAi construct for betasatellite) were challenged against Cotton leaf curl Khokran virus (CLCuKV) and Cotton leaf curl Multan betasatellite (CLCuMB). Reduced titer of the virus and betasatellite were detected through Southern blot hybridization. Significance of the study has been discussed.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02816-6.
Project description:Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman.
Project description:BackgroundCotton leaf curl disease, caused by single-stranded DNA viruses of the genus Begomovirus (family Geminiviridae), is a major constraint to cotton cultivation across Pakistan and north-western India. At this time only cotton varieties with moderate tolerance are available to counter the disease. microRNAs (miRNAs) are a class of endogenous small RNA molecules that play an important role in plant development, signal transduction, and response to biotic and a biotic stress. Studies have shown that miRNAs can be engineered to alter their target specificity. Such artificial miRNAs (amiRNAs) have been shown to provide resistance against plant-infecting viruses.ResultsTwo amiRNA constructs, based on the sequence of cotton miRNA169a, were produced containing 21 nt of the V2 gene sequence of Cotton leaf curl Burewala virus (CLCuBuV) and transformed into Nicotiana benthamiana. The first amiRNA construct (P1C) maintained the miR169a sequence with the exception of the replaced 21 nt whereas in the second (P1D) the sequence of the miRNA169a backbone was altered to restore some of the hydrogen bonding of the mature miRNA duplex. P1C transgenic plants showed good resistance when challenge with CLCuBV; plants being asymptomatic with low viral DNA levels. The resistance to heterologous viruses was lower and correlated with the numbers of sequence mismatches between the amiRNA and the V2 gene sequence. P1D plants showed overall poorer resistance to challenge with all viruses tested.ConclusionsThe results show that the amiRNA approach can deliver efficient resistance in plants against a monopartite begomoviruses and that this has the potential to be broad-spectrum, providing protection from a number of viruses. Additionally the findings indicate that the levels of resistance depend upon the levels of complementarity between the amiRNA and the target sequence and the sequence of the miRNA backbone, consistent with earlier studies.