Cotton leaf curl disease (CLCuD), a devastating disorder of cotton in Pakistan, is caused by a whitefly-transmitted begomovirus ( Cotton leaf curl virus ; CLCuV) that requires a satellite DNA β to cause disease symptoms (Mansoor et al ., 1993;Briddon et al ., 2001). CLCuD-resistant cotton varieties, in which no virus can be detected, have been developed through conventional breeding (Rahman et al ., 2002). During the 2001 growing season, symptoms of CLCuD were observed on all hitherto resistant varieties at Burewala, District Vehari, Pakistan, and by 2002 disease symptoms were seen throughout the district. To determine if a resistance-breaking strain of CLCuV had arisen, resistant and susceptible varieties were grown in the field at NIBGE (Faisalabad) and at the Cotton Research Station (Vehari). Plants of six commercial virus-resistant varieties (CIM 448, CIM 443, CIM 446, CIM 473, CIM 435 and FH 900) showed no disease symptoms at Faisalabad, while susceptible varieties S-12 and CIM70 had symptoms typical of CLCuD. At Vehari, plants of the same six resistant varieties showed between 15 and 50% infection, while the two susceptible varieties were all infected. Scions of CLCuD-affected resistant varieties, collected from Vehari, were grafted onto 10 plants of each resistant genotype at NIBGE. This resulted in disease symptoms on 20 -40% of plants, confirming a breakdown of resistance. To identity the resistance-breaking virus, nucleic acid was extracted from plants with and without symptoms collected at both sites. Samples were Southernblotted and probed with a biotinylated DNA A clone of CLCuV. The probe detected both the ss and ds DNA forms characteristic of begomoviruses, confirming the association of a begomovirus with the disease. Universal primers for DNA β of CLCuV were used to amplify DNA β from leaves with symptoms collected from resistant varieties in the Vehari area and the PCR product from one location was cloned in a T/A cloning vector (Fermentas).Since CLCuV DNA β is specific to CLCuV (Briddon et al ., 2003), a DNA β cloned from cotton plants of resistant varieties showing symptoms of CLCuD in the Burewala area was used as a disease-specific probe in Southern blot hybridizations. The probe hybridized only with DNA extracted from CLCuV affected cotton plants while no signal was detected from a tomato plant ( Lycopersicon esculentum ) that was previously shown to be associated with a DNA β distinct from that associated with CLCuV (Briddon et al ., 2003). Samples collected from both locations hybridized with this probe. A duplicate blot was probed with a previously reported CLCuV DNA β (Briddon et al ., 2001) and this resulted in a similar pattern of hybridization. Based on the data presented here, it was concluded that the plants of resistant varieties were infected with CLCuV (Briddon et al ., 2001). These results strongly suggest the emergence of a resistance-breaking strain of CLCuV in Pakistan. References

Geminiviruses are single-stranded DNA viruses that infect a broad range of dicotyledonous and monocotyledonous plants. Viruses of the family Geminiviridae have been classified into four genera based on host range and genome organization (30). Of these, the whitefly-transmitted geminiviruses (genus Begomovirus) are the largest group, with more than 100 virus species identified so far (8). Geminiviruses replicate in the nuclei of host cells by a rolling-circle replication mechanism (reviewed in reference 11). For the propagation of infection in the host, these viruses encode movement proteins that direct the viral genome to the cortical cytoplasm and across the barrier of the cell wall. For bipartite begomoviruses, this process requires two proteins, encoded by the component designated DNA B: the nuclear shuttle protein (NSP) and the cell-to-cell movement protein (MP). These proteins act cooperatively to move the viral DNA from its site of replication in the nucleus to the cytoplasm and into adjacent plant cells (32).The geminivirus MP and NSP recognize DNA in a form-and size-specific manner (12,29). The NSP shuttles newly replicated viral DNA between the nucleus and the cytoplasm (42). MP traps these NSP-viral DNA complexes in the cytoplasm and redirects them to and across the plant cell wall (24,43). In adjacent cells, the NSP-viral DNA complexes are released, and NSP targets the viral DNA to the nucleus to initiate new rounds of infection. This process of movement is highly regulated. Recently, an acetyltransferase has been isolated from Arabidopsis thaliana that interacts with the NSP encoded by the bipartite begomovirus Cabbage leaf curl virus (22). This protein regulates the nuclear export of the viral DNA and is highly conserved in plants.

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The hypersensitive response (HR) is a common feature of plant disease resistance reactions and a type of programmed cell death (PCD). Many pathogens are able to modulate pathways involved in cell death. In contrast to animal viruses, inhibitors of PCD activity have not been identified for plant-infecting viruses. Previously, we have reported that the nuclear shuttle protein (NSP) of Tomato leaf curl New Delhi virus (ToLCNDV) induces an HR in Nicotiana tabacum and Lycopersicon esculentum plants when expressed under the control of the Cauliflower mosaic virus 35S promoter. However, HR is not evident in plants infected with ToLCNDV, suggesting that the virus encodes a factor (or factors) that counters this response. Analysis of all ToLCNDV-encoded genes pinpointed the transcriptional activator protein (TrAP) as the factor mediating the anti-HR effect. Deletion mutagenesis showed the central region of TrAP, containing a zinc finger domain and nuclear localization signal, to be important in inhibiting the HR. These results demonstrate that TrAP counters HR-induced cell death, the first such activity identified for a plant-infecting virus.

Chilli leaf curl disease is an important constraint to production of chilli peppers (Capsicum annum L.) across the Indian subcontinent and is caused by begomoviruses in association with betasatellites. Betasatellites are symptom modulating single-stranded DNA satellites associated with the majority of monopartite begomoviruses and are responsible for diseases of many crops including chilli peppers. Here, we have studied the diversity of a betasatellite of chillies in the Punjab and North Western Frontier Province of Pakistan. Sequence analysis of thirteen full-length clones showed that a single species of satellite, Chilli leaf curl betasatellite (ChLCB), is prevalent in chilli throughout the surveyed region. ChLCB has only been identified in Pakistan and infecting chilli. It thus probably represents a chilli adapted satellite. The ChLCBs fall into two groups, the first occurs in the less intensively cultivated north and shows a high level of sequence diversity (relative to the second group), while the second occurs in the southern cotton-growing areas and shows little sequence diversity. Between these two areas there is a region of overlap where both ChLCB types occur. The phylogeographic segregation of ChLCB we attribute to geographic isolation (in the north), leading to divergence and intensive cultivation (in the south), leading to homogenization. However, the lack of diversity (only a single species) bodes well for the prospects of achieving resistance to the disease by either conventional or pathogen-derived strategies.