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Cotton leaf curl disease (CLCuD) is one of the major limiting factors affecting cotton production in Pakistan for the last three decades. The disease is caused by begomoviruses of the family Geminiviridae. RNA interference (RNAi) is a promising tool that has been proved effective against several pathogens. Using RNAi, different genomic regions of geminiviruses have been targeted to attain sustainable resistance. However, the silencing of the transgene upon virus infection is a limiting factor. Here, we have developed for the first time an amplicon-based RNAi construct to target βC1 gene of betasatellite associated with cotton leaf curl begomoviruses. In addition to producing short interfering (si) RNAs, Rep-based activation or looping out of the construct induced upon virus infection produces multiple copies of transgene that results in accumulation of defective molecules of betasatellite. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Transgenic Nicotiana benthamiana plants having RCβ (RNAi construct for betasatellite) were challenged against Cotton leaf curl Khokran virus (CLCuKV) and Cotton leaf curl Multan betasatellite (CLCuMB). Reduced titer of the virus and betasatellite were detected through Southern blot hybridization. Significance of the study has been discussed. [ABSTRACT FROM AUTHOR]

Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan) Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world. [ABSTRACT FROM AUTHOR]