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Engineering tolerance to CLCuD in transgenic Gossypium hirsutum cv. HS6 expressing Cotton leaf curl Multan virus-C4 intron hairpin.

ABSTRACT: Cotton leaf curl disease (CLCuD), caused by begomoviruses in combination with betasatellite molecule, has adversely affected cotton industry of Indian subcontinent. To devise a CLCuD-control strategy, RNAi-mediated approach was followed in this study. Gossypium hirsutum cv. HS6 plants were transformed with intron-hairpin RNAi (ihpRNAi-C4) construct carrying silencing suppressor C4 gene of Cotton leaf curl Multan virus (CLCuMuV). Efficacy of the construct in imparting CLCuD resistance was evaluated in transgenic (T₀, T₁) cotton lines. Accumulation of CLCuMuV/betasatellite and attenuation of CLCuD symptoms in the transgenic lines were monitored at different times interval after virus inoculation. Northern hybridization revealed the expression of C4-gene derived siRNA. Expression of the ihpRNAi transcript was recorded higher in transgenic lines expressing siRNA which supposedly targeted the C4 gene. A significant delay in detection of virus as well as betasatellite was observed in the transgenic lines. At 30 days post inoculation (dpi), none of the lines tested positive. At 45 dpi, however, it could be detected in few lines having much lower titre as compared to non-transformed control plants. Notably, till 60 dpi, no significant progression of the virus/betasatellite DNA was observed and the plants did not exhibit any characteristic CLCuD symptoms. A tolerance phenomenon leading to escape of CLCuD symptoms in the transformed cotton was described.

SUBMITTER: Baig MS

PROVIDER: S-EPMC8266814 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWhitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies.ResultsThe major transcripts of CLCuBuV and its associated betasatellite (CLCuMuB) from infected Nicotiana benthamiana plants have been determined. Two complementary-sense transcripts of ~1.7 and ~0.7 kb were identified for CLCuBuV. The ~1.7 kb transcript appears similar in position and size to that of several begomoviruses and likely directs the translation of C1 and C4 proteins. Both complementary-sense transcripts can potentially direct the translation of C2 and C3 proteins. A single virion-sense transcript of ~1 kb, suitable for translation of the V1 and V2 genes was identified. A predominant complementary-sense transcript was also confirmed for the betasatellite.ConclusionsOverall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein.

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Engineering tolerance to CLCuD in transgenic Gossypium hirsutum cv. HS6 expressing Cotton leaf curl Multan virus-C4 intron hairpin.

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