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Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

ABSTRACT: Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.

SUBMITTER: Ismayil A

PROVIDER: S-EPMC6133388 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Ismayil Asigul A Haxim Yakupjan Y Wang Yunjing Y Li Huangai H Qian Lichao L Han Ting T Chen Tianyuan T Jia Qi Q Yihao Liu Alexander A Zhu Songbiao S Deng Haiteng H Gorovits Rena R Hong Yiguo Y Hanley-Bowdoin Linda L Liu Yule Y

PLoS pathogens 20180829 8

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, ...[more]

PMID: 30157283

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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:Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.

Dataset Information

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Publications

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Similar Datasets

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