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Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman.

ABSTRACT: During a survey in February 2016, leaf curl disease symptoms were witnessed in Cucurbita maxima plants in Al-Batina commercial farm in Oman. Symptoms exhibited were characteristic of begomovirus infection as leaf curling, yellowing, and color breaking followed by mosaic pattern. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. Rolling circle amplification (RCA) was employed to characterize the unknown causal agent of C. maxima disease. In molecular identification RCA produced nearly 2.8 and 1.4 kb DNA molecules corresponding to begomovirus and satellite molecules, cloned and sequenced them. In Blast, species demarcation tool and phylogenetic analysis revealed the begomovirus and satellite isolates were determined as Chilli leaf curl virus (ChLCV) and tomato leaf curl betasatellite (ToLCB). In biological analysis by agrobacterium mediated inoculation, ChLCV displayed upward leaf curling and vein swelling symptoms in Nicotiana benthamiana plants; however, in presence of ToLCB enhanced downward leaf curling and crumpling symptoms were revealed. This study provides the first evidence that ChLCV and ToLCB caused leaf curl disease of C. maxima in Oman.

SUBMITTER: Shahid MS

PROVIDER: S-EPMC7459049 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn 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.MethodsCircular 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.ResultsThe 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.ConclusionsThe 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:BackgroundWhitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies.ResultsThe major transcripts of CLCuBuV and its associated betasatellite (CLCuMuB) from infected Nicotiana benthamiana plants have been determined. Two complementary-sense transcripts of ~1.7 and ~0.7 kb were identified for CLCuBuV. The ~1.7 kb transcript appears similar in position and size to that of several begomoviruses and likely directs the translation of C1 and C4 proteins. Both complementary-sense transcripts can potentially direct the translation of C2 and C3 proteins. A single virion-sense transcript of ~1 kb, suitable for translation of the V1 and V2 genes was identified. A predominant complementary-sense transcript was also confirmed for the betasatellite.ConclusionsOverall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein.

Project description:BackgroundTomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels.ResultsA hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants.ConclusionsThese results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

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Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman.

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