Expression of full-length and truncated Rep genes from Mungbean yellow mosaic virus-Vigna inhibits viral replication in transgenic tobacco

Abstract

Mungbean yellow mosaic virus-Vigna (MYMV-Vig) is a bipartite geminivirus that causes a severe yellow mosaic disease in blackgram. An assay was developed to study MYMV-Vig replication by agroinoculation of tobacco leaf discs with partial dimers of the virus. This assay, in a non-host model plant, was used to evaluate pathogen-derived resistance contributed by MYMV-Vig genes in transgenic plants. Viral DNA accumulation was optimum in tobacco leaf discs cultured for 10 days after infection with Agrobacterium tumefaciens strain Ach5 containing partial dimers of both DNA A and DNA B of MYMV-Vig. Transgenic tobacco plants with MYMV-Vig genes for coat protein (CP), replication-associated protein (Rep)-sense, Rep-antisense, truncated Rep (T-Rep), nuclear shuttle protein (NSP) and movement protein (MP) were generated. Leaf discs from transgenic tobacco plants, harbouring MYMV-Vig genes, were agroinoculated with partial dimers of MYMV-Vig and analyzed for viral DNA accumulation. The leaf discs from transgenic tobacco plants harbouring CP and MP genes supported the accumulation of higher levels of MYMV-Vig DNA. However, MYMV-Vig accumulation was inhibited in one transgenic plant harbouring the Rep-sense gene and in two plants harbouring the T-Rep gene. Northern analysis of these plants revealed a good correlation between expression of Rep or T-Rep genes and inhibition of MYMV-Vig accumulation.

Antibegomoviral activity of the agrobacterial virulence protein VirE2

“…Alternatively, we found that tobacco (cv. Wisconsin 38) leaf discs, agroinoculated with the partial dimers of MYMV DNA A and DNA B, replicated and accumulated MYMV DNA A [29]. We raised transgenic tobacco plants with the candidate genes and used the tobacco leaf disc agroinoculation assay to evaluate the effect of transgenes on MYMV DNA accumulation.…”

“…Three transgenic TrAP-DAD-tobacco plants which accumulated varying levels of the TrAP-DAD transcript and two VirE2-transgenic tobacco plants which complemented a virE2 mutation in Agrobacterium were evaluated for inhibition of MYMV DNA replication using the leaf disc agroinoculation assay [29]. The plants which accumulated higher levels of the TrAP-DAD and virE2 transcripts displayed a pronounced reduction in MYMV DNA accumulation.…”

Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences

“…Brunetti et al [36] also reported resistance in tomato plants against TYLCV using truncated Rep protein. Deletion of three motifs (conserved in all geminiviral AL1/AC1 protein) at N-and C-terminus may lead to the abolition of the role of DNA cleavage, ligation, ATP hydrolysis, and cell cycle regulation that will ultimately affect viral replication [7]. Out of four 5 0 AtC1 plant lines expressing first 210 amino acids of N-terminus, only one line gave reliable resistance.…”

“…Transgenic expression of pathogen-derived sequences has variously been explored and exploited the general idea that the expression of virus-derived selective sequences may interfere with the viral life cycle. The transgenic sequence can act via a protein-mediated mechanism or by post-transcriptional gene silencing (PTGS) resulting in virus tolerance [7][8][9]. Expression of Rep in sense orientation inhibited African cassava mosaic virus (ACMV) replication [10].…”

Application of molecular antiviral compounds: novel approach for durable resistance against geminiviruses

“…Collapse in degree of geminivirus resistance in various transgenic plants has also been reported. Failure or limited effectiveness of the RNAi based strategies has been highlighted in the case of MSV [33] and Mungbean yellow mosaic virus [34]. In these cases, full-length or truncated antisense Rep gene constructs were found to be ineffective in providing resistance.…”