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Research article2012Peer reviewed

Effects of the mutation of selected genes of Cotton leaf curl Kokhran virus on infectivity, symptoms and the maintenance of Cotton leaf curl Multan betasatellite

Iqbal, Zafar; Sattar, Muhammad Naeem; Kvarnheden, Anders; Mansoor, Shahid; Briddon, Rob


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 infectivityand symptoms in Nicotiana benthamiana were investigated. Each mutation introduced a premature stopcodon which would lead to premature termination of translation of the gene. Mutation of the CP geneabolished infectivity. However, transient expression of the CLCuKoV CP gene under the control of theCauliflower mosaic virus 35S promoter (35S-KoCP), at the point of inoculation, led to a small number ofplants in which viral DNA could be detected by PCR in tissues distal to the inoculation site. Mutations ofthe V2, C2 and C4 genes reduced infectivity. The V2 and C2 mutants did not induce symptoms, whereasthe C4 mutation was associated with attenuated symptoms. Infections of plants with the C4 mutant wereassociated with viral DNA levels equivalent to the wild-type virus, whereas viral DNA levels for the V2mutant were low, detectable by Southern blot hybridisation, and for the C2 mutant were detectable onlyby PCR. Significantly, transient expression of the CLCuKoV C2 gene at the point of inoculation, raisedvirus DNA levels in tissues distal to the inoculation site such that they could be detected by Southernhybridisation, although they remained at well below the levels seen for the wild-type virus, but reducedthe infectivity of the virus. These findings are consistent with earlier mutation studies of monopartitebegomoviruses and our present knowledge concerning the functions of the four genes suggesting that theCP 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 suggestthat the V2, C2 and C4 may be pathogenicity determinants. Additionally the effects of the mutations ofCLCuKoV genes on infections of the virus in the presence of its cognate betasatellite, Cotton leaf curl Multanbetasatellite (CLCuMuB), were investigated. Mutation of the C4 gene had no effect on maintenanceof the betasatellite, although the betasatellite enhanced symptoms. Inoculation of the C2 mutant withCLCuMuB raised the infectivity of the virus to near wild-type levels, although the numbers of plants inwhich the betasatellite was maintained was reduced, in comparison to wild-type virus infections withCLCuMuB, and viral DNA could not be detected by Southern hybridisation. Transient expression of theC2 gene at the point of inoculation raised virus DNA levels in tissues distal to the inoculation site but alsoreduced 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 ofplants was the satellite maintained and infections were non-symptomatic. Although inoculation of theCP mutant with CLCuMuB did not restore infectivity, co-inoculation with 35S-KoCP increased the numberof plants in which the virus could be detected, in comparison to plants inoculated with the mutantand 35S-KoCP, and also resulted in two plants (out of 15 inoculated) in which the betasatellite could bedetected by PCR. This indicates that the V2, C2 and almost certainly the CP are important for the maintenanceof betasatellites by monopartite begomoviruses. The significance of these findings is discussed.


Begomovirus Betasatellite Mutation Symptoms Movement Replication

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Virus Research
2012, Volume: 169, number: 1, pages: 107-116

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