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Cotton crop has been severely affected by multiple begomoviruses in Pakistan and India. In our previous study, we found okra enation leaf curl virus (OELCuV), cotton leaf curl Multan betasatellite (CLCuMuB) and cotton leaf curl Multan alphasatellite (CLCuMuA) infecting cotton in Pakistan. The current study was designed to investigate the infectivity of OELCuV and its ability to trans-replicate non-cognate CLCuMuB. Agro-infectious clones containing the partial tandem repeats of OELCuV and CLCuMuB were constructed and the infectivity assays were carried out through Agrobacterium mediated transformation in the model host species Nicotiana benthamiana under controlled conditions. The results showed that in the inoculated plants OELCuV alone can cause downward curling and yellowing of leaves with thickened veins. However, when co-inoculated with the non-cognate CLCuMuB it could functionally trans-replicate CLCuMuB resulting in a more severe phenotype. The expression of Pre-coat/V2 protein in the N. benthamiana plants through the potato virus X (PVX) system caused localized cell death after severe leaf curling in the infiltrated leaves. The tissue tropism of the virus was associated with the systemic development of a hypersensitive response (HR), which ultimately lead to the plant death. The results indicated the involvement of V2 protein in the pathogenicity of OELCuV and its ability to trigger the host defense machinery. This study also demonstrated the ability of OELCuV to trans-replicate CLCuMuB resulting in typical leaf curl disease symptoms in N. benthamiana., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Okra leaf curl disease (OLCD) is the most important viral disease of okra in West Africa. In this study, a complex of begomoviruses and associated DNA satellites were identified in symptomatic okra plants from southwestern Cameroon. Sequence analyses showed that two of the plants (Lik1 and Njo5) were infected with a begomovirus being a recombinant of cotton leaf curl Gezira virus (CLCuGeV) and okra yellow crinkle virus (OYCrV). The recombinant genome shared highest nucleotide identity with isolates of CLCuGeV at 87.8% and is therefore considered to be member of a new begomovirus species, Okra leaf curl Cameroon virus (OLCuCMV). One plant (Mue5) was infected by a begomovirus with 95.8% nucleotide identy to CLCuGeV, while in the plants Lik1, Mue1 and Njo5, a begomovirus was identified showing highest nucleotide identity at 93.7% with OYCrV. The nucleotide comparisons and phylogenetic analyses suggest that these isolates represent new Cameroonian strains of CLCuGeV and OYCrV (CLCuGeV-CM and OYCrV-CM). Mixed infection of OLCuCMV and OYCrV-CM was found in two of the plants. A betasatellite and two divergent alphasatellites were also associated with the begomoviruses. The betasatellite was identified as cotton leaf curl Gezira betasatellite (CLCuGeB) with the highest nucleotide identity at 93.3% to other African isolates of CLCuGeB. The alphasatellites, herein named Alpha-1 and Alpha-2, shared 97.3% and 95.2% identity, respectively, with cotton leaf curl Gezira alphasatellite (CLCuGeA) and okra leaf curl Burkina Faso alphasatellite (OLCuBFA). These collective results emphasize the extent of diversity among okra-infecting begomovirus-satellite complexes in western Africa., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cotton leaf curl Kokhran virus (CLCuKoV) is a cotton-infecting monopartite begomovirus (family Geminiviridae). The effects of mutation of the coat protein (CP), V2, C2 and C4 genes of CLCuKoV on infectivity and symptoms in Nicotiana benthamiana were investigated. Each mutation introduced a premature stop codon which would lead to premature termination of translation of the gene. Mutation of the CP gene abolished infectivity. However, transient expression of the CLCuKoV CP gene under the control of the Cauliflower mosaic virus 35S promoter (35S-Ko(CP)), at the point of inoculation, led to a small number of plants in which viral DNA could be detected by PCR in tissues distal to the inoculation site. Mutations of the V2, C2 and C4 genes reduced infectivity. The V2 and C2 mutants did not induce symptoms, whereas the C4 mutation was associated with attenuated symptoms. Infections of plants with the C4 mutant were associated with viral DNA levels equivalent to the wild-type virus, whereas viral DNA levels for the V2 mutant were low, detectable by Southern blot hybridisation, and for the C2 mutant were detectable only by PCR. Significantly, transient expression of the CLCuKoV C2 gene at the point of inoculation, raised virus DNA levels in tissues distal to the inoculation site such that they could be detected by Southern hybridisation, although they remained at well below the levels seen for the wild-type virus, but reduced the infectivity of the virus. These findings are consistent with earlier mutation studies of monopartite begomoviruses and our present knowledge concerning the functions of the four genes suggesting that the CP is essential for long distance spread of the virus in plants, the C4 is involved in modulating symptoms, the C2 interferes with host defence and the V2 is involved in virus movement. The results also suggest that the V2, C2 and C4 may be pathogenicity determinants. Additionally the effects of the mutations of CLCuKoV genes on infections of the virus in the presence of its cognate betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB), were investigated. Mutation of the C4 gene had no effect on maintenance of the betasatellite, although the betasatellite enhanced symptoms. Inoculation of the C2 mutant with CLCuMuB raised the infectivity of the virus to near wild-type levels, although the numbers of plants in which the betasatellite was maintained was reduced, in comparison to wild-type virus infections with CLCuMuB, and viral DNA could not be detected by Southern hybridisation. Transient expression of the C2 gene at the point of inoculation raised virus DNA levels in tissues distal to the inoculation site but also reduced the infectivity of the virus and the numbers of plants in which the betasatellite was maintained. CLCuMuB restored the infectivity of the V2 mutant to wild-type levels but only in a small number of plants was the satellite maintained and infections were non-symptomatic. Although inoculation of the CP mutant with CLCuMuB did not restore infectivity, co-inoculation with 35S-Ko(CP) increased the number of plants in which the virus could be detected, in comparison to plants inoculated with the mutant and 35S-Ko(CP), and also resulted in two plants (out of 15 inoculated) in which the betasatellite could be detected by PCR. This indicates that the V2, C2 and almost certainly the CP are important for the maintenance of betasatellites by monopartite begomoviruses. The significance of these findings is discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)