G5, a Phage Single-Stranded DNA-Binding Protein, Fused with a Nuclear Localization Signal, Attenuates Symptoms and Reduces Begomovirus-Betasatellite Accumulation in Transgenic Plants

Abstract

Cotton leaf curl disease is caused by several monopartite begomoviruses and is the major threat to cotton production in the Indian subcontinent. The disease has been shown to be associated with four distinct species, including Cotton leaf curl Kokhran virus (CLCuKoV), and a specific betasatellite-Cotton leaf curl Multan betasatellite (CLCuMuB). Transgenic Nicotiana benthamiana plants were produced which constitutively express the Escherichia coli phage M13 encoded, sequence nonspecific single-stranded (ss) DNA-binding protein, G5 alone and fused with the maize opaque-2 nuclear localization signal (NLS), to evaluate resistance against CLCuKoV-CLCuMuB. Transgenic plants expressing only G5 performed poorly exhibiting symptoms of infection and high virus DNA levels upon inoculation with CLCuKoV and CLCuKoV with CLCuMuB. In contrast, plants transformed with G5 fused to the NLS developed mild symptoms and showed a reduction in virus and betasatellite DNA levels in comparison to nontransformed plants. The results show that G5 may be useful in developing broad-spectrum resistance against ssDNA viruses.

Tolerance to tomato leaf curl New Delhi begomovirus in transgenic Nicotiana benthamiana expressing the phage M13 gene 5 (G5), an ssDNA binding protein

“…Our study showed that transgenic N. benthamiana lines expressing G5fused with the maize opaque-2 nuclear localization signal (NLS) have tolerance to ToLCNDV. This extends earlier work on G5, which showed this to provide tolerance to infection by a monopartite betasatellite-associated begomovirus (Rasool et al 2016), and indicates that G5 may be a useful tool in generating crops with broad spectrum resistance to begomoviruses.…”

“…Overall, these results are in agreement with an earlier study, using the same transgenic NLS-G5 N. benthamiana lines, which showed resistance to the monopartite begomovirus cotton leaf The molecular detection show PCR and Southern hybridization detection results. Southern blots showed no hybridization (N) from non-symptomatic plants, low hybridization (L) resulted from mild symptomatic plants and high levels of hybridization (H) from severely symptomatic plant samples curl Kokhran virus (CLCuKoV) in the presence and absence of its cognate betasatellite (Rasool et al 2016). In the previous study, a significant proportion of inoculated plants did not develop symptoms as found in our work.…”

“…The production of transgenic N. benthamiana plants (lines2 and 4 of the T 2 generation) using an expression vector pJIT60 under the control of the cauliflower mosaic virus (CaMV) 35S promoter expressing NLS-G5 has been described previously (Rasool et al 2016). Bottom leaves of plants were challenged at the four-leaf stage with ToLCNDV by Agrobacterium-mediated inoculation as described previously (Saeed 2008;Padidam et al 1995).…”

Transgenic Expression of Synthetic Coat Protein and Synthetic Replication Associated Protein Produces Mild Symptoms and Reduce Begomovirus-Betasatellite Accumulation in Nicotiana benthamiana

“…The coat protein (CP) sequence of CLCuKoV-Bu was assembled and compared with the (accession number HF549183) CPs of other begomoviruses in the database, using BLAST (Mullineaux et al, 1988). The detailed mechanism of point mutations introduced in the CP sequence has been mentioned in Rasool et al (2016a). The designed mutated CP construct was codon optimized for Gossypium hirsutum (http://www.…”