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Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India.

ABSTRACT: Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for three years and subjected to DNA isolation, followed by rolling circle amplification (RCA), cloning, and DNA sequencing of apparently full-length begomoviral genomes and associated betasatellites and alphasatellites. Among the thirteen CLCuD-begomoviral genomes recovered, eight were identified as Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra), one as -Pakistan (PK) and another as -Faisalabad (Fai), whereas, three were as Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu), indicating that CLCuMuV-Ra was the most prevalent begomovirus species. Five of the eight CLCuMuV-Ra sequences were found to be recombinants. The CLCuMuV-Ra- associated satellites consisted of Cotton leaf curl Multan betasatellite (CLCuMB), and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). The second most abundant helper virus species, CLCuKoV-Bu, was associated with CLCuMB and GDarSLA.

SUBMITTER: Biswas KK

PROVIDER: S-EPMC7176085 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India.

Biswas Kajal Kumar KK Bhattacharyya Utpal Kumar UK Palchoudhury Supratik S Balram Nenavath N Kumar Anil A Arora Rupesh R Sain Satish Kumar SK Kumar Pradeep P Khetarpal Ravi K RK Sanyal Amitava A Mandal Pranab Kumar PK

PloS one 20200422 4

Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for ...[more]

PMID: 32320461

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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.

Project description:BackgroundCotton leaf curl disease (CLCuD), caused by begomoviruses in association with satellite molecules, is a major threat to cotton production causing enormous losses to cotton crop in most of the cotton growing countries including Indian subcontinent. In this study, isolates of begomovirus and satellite molecules associated with CLCuD were collected from North India (Haryana, New Delhi). They were amplified employing rolling circle replication mechanism, cloned, sequenced and, their phylogenetic and recombination analysis was performed.ResultsThe five Cotton leaf curl Multan virus (CLCuMuV) isolates investigated in this study showed monopartite organization of the genome typical of Old World begomoviruses. Nucleotide sequence analyses assigned them as the strains of CLCuMuV and were designated as CLCuMuV-SR13, CLCuMuV-SR14, CLCuMuV-ND14, CLCuMuV-ND15 and CLCuMuV-SR15. The genome of CLCuMuV-SR13 shared a highest level of nucleotide sequence identity (98%) with CLCuMuV (JN678804), CLCuMuV-SR14 and CLCuMuV-SR15 exhibited 96% with CLCuMuV (KM096471), while isolates CLCuMuV-ND15 and CLCuMuV-SR15 revealed 96% sequence identity with CLCuMuV (AY765253). The four betasatellite molecules investigated in this study shared 95-99% nucleotide sequence identity with Cotton leaf curl Multan betasatellite (CLCuMB) from India. The betasatellite molecules were designated as CLCuMB-SR13, CLCuMB-SR14, CLCuMB-ND14 and CLCuMB-ND15. Alphasatellite molecules in this study, designated as GLCuA-SR14, GLCuA-ND14 and GLCuA-SR15, revealed 98% identity with Guar leaf curl alphasatellite (GLCuA) reported from Pakistan.ConclusionThe phylogenetic and recombination studies concluded that the isolates of CLCuMuV genomes undertaken in this study have a potential recombinant origin. Remarkably, significant recombination was detected in almost all the genes with contribution of Cotton leaf curl Kokhran Virus (CLCuKoV) in IR, V1, V2, C1, C4 and C5 regions and of CLCuMuV in C2 region of CLCuMuV-SR14. CLCuKoV also donated in C2, C3 regions of CLCuMuV-ND14; V1, V2, C2 and C3 regions of CLCuMuV-ND15 and C1 of CLCuMuV-SR15. Altogether, these observations signify the uniqueness in Indian CLCuMuV isolates showing contribution of CLCuKoV in all the genes. An interesting observation was frequent identification of GLCuA in CLCuD leaf samples.

Dataset Information

Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India.

Publications

Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India.

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