Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions.

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

Development, Design, and Application of Efficient siRNAs Against Cotton Leaf Curl Virus-Betasatellite Complex to Mediate Resistance Against Cotton Leaf Curl Disease.

Cotton leaf curl disease (CLCuD), caused by the Cotton leaf curl virus, is one of the most irrepressible diseases in cotton due to high recombination in the virus. RNA interference (RNAi) is widely used as a biotechnological approach for sequence-specific gene silencing guided by small interfering RNAs (siRNAs) to generate resistance against viruses. The success of RNAi depends upon the fact that the target site of the designed siRNA must be conserved even if the genome undergoes recombination. Thus, the present study designs the most efficient siRNA against the conserved sites of the Cotton leaf curl Multan virus (CLCuMuV) and the Cotton leaf curl Multan betasatellite (CLCuMB). From an initial prediction of 9 and 7 siRNAs against CLCuMuV and CLCuMB, respectively, the final selection was made for 2 and 1 siRNA based on parameters such as no off-targets, good GC content, high validity score, and targeting coding region. The target sites of siRNA were observed to lie in the AC3 and an overlapping region of AC2-AC1 of CLCuMuV and ßC1 of CLCuMB; all target sites showed a highly conserved nature in recombination analysis. Docking the designed siRNAs with the Argonaute-2 protein of Gossypium hirsutum showed stable binding. Finally, BLASTn of siRNA-target positions in genomes of other BGVs indicated the suitability of designed siRNAs against a broad range of BGVs. The designed siRNAs of the present study could help gain complete control over the virus, though experimental validation is highly required to suggest predicted siRNAs for CLCuD resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01191-z.