Virus Research 169 (2012) 107–116 Contents lists available at SciVerse ScienceDirect Virus Research jo u r n al hom epa ge: www.elsevier.com/locate/virusres 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 Zafar Iqbal a , M. Naeem Sattar b , Anders Kvarnheden b , Shahid Mansoor a , Rob W. Briddon a,∗ a Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan b Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Linnean Centre of Plant Biology in Uppsala, Swedish University of Agricultural Sciences, Uppsala, Sweden a r t i c l e i n f o Article history: Received 5 April 2012 Received in revised form 13 July 2012 Accepted 17 July 2012 Available online 31 July 2012 Keywords: Begomovirus Betasatellite Mutation Symptoms Movement Replication a b s t r a c t Cotton leaf curl Kokhran virus (CLCuKoV) is a cotton-infecting monopartite begomovirus (family Gemi- niviridae). 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 Mul- tan 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 num- ber 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 main- tenance of betasatellites by monopartite begomoviruses. The significance of these findings is discussed. © 2012 Elsevier B.V. All rights reserved. ∗ Corresponding author. Tel.: +92 41 2651475. E-mail addresses: zafariqbal2009@gmail.com (Z. Iqbal), naeem.sattar@slu.se (M.N. Sattar), anders.kvarnheden@slu.se (A. Kvarnheden), shahidmansoor7@gmail.com (S. Mansoor), rob.briddon@gmail.com (R.W. Briddon). 1. Introduction Viruses of the family Geminiviridae have small, single-stranded (ss)DNA genomes that are encapsidated in characteristic twinned quasi-icosahedral particles and are ascribed to one of the four 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.07.016