Evaluation of a single-stranded DNA-binding protein (VirE2) and a synthetic replication-associated protein (Repsyn130) to control cotton leaf curl disease in Nicotiana benthamiana

Abstract

Cotton leaf curl disease (CLCuD) is the most important biotic constraint to cotton production on the subcontinent and has a direct impact on the economy of Pakistan, since it is the major cash crop. The disease is caused by a complex of begomoviruses, the most important of which at this time is Cotton leaf curl Burewala virus (CLCuBuV), associated with a disease specific, symptom determining satellite – Cotton leaf curl Multan betasatellite (CLCuMuB). Begomoviruses (family Geminiviridae) are plant-infecting viruses with genomes consisting of single-stranded (ss) DNA. Begomoviruses are transmitted by the whitefly Bemisia tabaci and cause significant crop losses worldwide. Begomoviruses evolve rapidly by componennt reassortment and recombination. Sources of host-plant resistance against begomoviruses are limited and genetic engineering provides a possible means of obtaining crop varieties with resistance. Genetically engineered resistance is of two general types; pathogen-derived-resistance (PDR) and non-pathogen-derived resistance (NPDR). PDR utilizes sequences derived from the pathogen to induce resistance. At this time most reported PDR resistance is based on initiating gene silencing (GS; also known as RNA interference - a natural defense mechanism against foreign nucleic acids). Since GS is a homology-based process, virus-derived transgenes are prone to silencing upon virus infection. NPDR, in contrast, involves the use of non-viral sequences and is less widely investigated than silencing-based PDR approaches. The study described here was designed to investigate both PDR and NPDR based strategies to the control of begomoviruses, with special emphasis to CLCuD. The PDR approach used a synthetic gene based on the sequence of the CLCuBuV-encoded replication-associated protein (Rep) gene designed to circumvent the issue of GS. An artificial gene of 390 nucleotides, encoding the N-terminal 130 amino acids of the CLCuBuV-Rep (Repsyn130) was designed to avoid GS by introducing silent point mutations. The NPDR approach used a single- stranded DNA-binding protein (SSB), VirE2, from Agrobacterium tumefaciens, The VirE2 gene was PCR amplified from A. tumefaciens strain C58. The two genes were each cloned under the control of a constitutive promoter in a binary vector. An initially assessment of the constructs for the potential to deliver resistance against begomoviruses used an Agrobacterium-mediated transient assay; in which the gene expression constructs were co-inoculated with infectious clones of selected begomoviruses and betasatellites. The transient assay showed Repsyn130 to provide good resistance against Cotton leaf curl Kokhran virus (CLCuKoV; a virus closely related to CLCuBuV)/Cotton leaf curl Multan betasatellite (CLCuMuB). Two types of plants were evident following inoculation. The first showed no symptoms, whereas the second developed only mild symptoms and many plants recovered. In both cases plants contained only low concentration of viral DNA. The Repsyn130 construct yielded poor resistance against Tomato leaf curl New Delhi virus (ToLCNDV; a virus only distantly related to CLCuBuV). Inoculated plants all showed severe symptoms, although symptoms took longer to appear than for inoculations without Repsyn130. Co-inoculation of either CLCuKoV/CLCuMuB or ToLCNDV with the construct for expression of VirE2 resulted in plants that showed a delay in the onset of symptoms, mild symptoms and a reduction in viral DNA. These results suggested that both strategies could interfere with virus infection. Transgenic N. benthamiana lines were produced using the VirE2 and Repsyn130 constructs by the Agrobacterium-mediated method. Selected lines were screened for resistance by inoculation with CLCuBuV and CLCuMuB. For N. benthamiana lines harboring the Repsyn130 construct, plants showed two types of response following inoculation. The first group of plants did not display symptoms of infection, although viral DNA could be detected by PCR but not by Southern hybridization, indicating that virus titer were very low in the plants. The second group of plants developed mild symptoms of infection, from which some plants recovered, and viral DNA could be detected by Southern hybridization, but at a titer lower than that typical for infections of non-transgenic N. benthamiana plants. N. benthamiana lines harbouring the VirE2 did not develop symptoms, following inoculation with CLCuBuV/CLCuMuB, and virus could be detected by PCR but not Southern hybridization. The results of the study showed that both approaches investigated provide a level of resistance to virus infection but neither provides immunity. The findings are discussed in relation to the proposed mechanism of action of each of the proteins and their possible use in reducing losses due to begomovirus infections of crop plants is outlined.