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Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

ABSTRACT: The replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13-28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38-40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners.

SUBMITTER: Shakir S

PROVIDER: S-EPMC7778653 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:RNA silencing is a conserved mechanism in plants that targets viruses. Viral small RNAs (vsiRNAs) can be generated from viral double-stranded RNA replicative intermediates within the infected host, or from host RNA-dependent RNA polymerases activity on viral templates. The abundance and profile of vsiRNAs in viral infections have been reported previously. However, the involvement of vsiRNAs during infection of the Geminiviridae family member cotton leaf curl virus (CLCuD), which causes significant economic losses in cotton growing regions, remains largely uncharacterized. Cotton leaf curl Multan virus (CLCuMuV) associated with a betasatellite called Cotton leaf curl Multan betasatellite (CLCuMuB) is a major constraint to cotton production in South Asia and is now established in Southern China. In this study, we obtained the profiles of vsiRNAs from CLCuMV and CLCuMB in infected upland cotton (Gossypium hirsutum) plants by deep sequencing. Our data showed that vsiRNA that were derived almost equally from sense and antisense CLCuD DNA strands accumulated preferentially as 21- and 22-nucleotide (nt) small RNA population and had a cytosine bias at the 5'-terminus. Polarity distribution revealed that vsiRNAs were almost continuously present along the CLCuD genome and hotspots of sense and antisense strands were mainly distributed in the Rep proteins region of CLCuMuV and in the C1 protein of CLCuMuB. In addition, hundreds of host transcripts targeted by vsiRNAs were predicted, many of which encode transcription factors associated with biotic and abiotic stresses. Quantitative real-time polymerase chain reaction analysis of selected potential vsiRNA targets showed that some targets were significantly down-regulated in CLCuD-infected cotton plants. We also verified the potential function of vsiRNA targets that may be involved in CLCuD infection by virus-induced gene silencing (VIGS) and 5'-rapid amplification of cDNA end (5'-RACE). Here, we provide the first report on vsiRNAs responses to CLCuD infection in cotton.

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Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

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