Cowpea (Vigna unguiculata L. [Walp.]) in one of the main grain legumes contributing to food security and poverty alleviation in Sub-Saharan Africa. To control the highly damaging legume pod borer Maruca vitrata F., tr...Cowpea (Vigna unguiculata L. [Walp.]) in one of the main grain legumes contributing to food security and poverty alleviation in Sub-Saharan Africa. To control the highly damaging legume pod borer Maruca vitrata F., transgenic cowpea lines expressing the insecticidal Cry1Ab Bt protein were developed. In this study, we evaluated the impact of Cry1Ab transgene expression on the susceptibility of four cowpea lines (named IT97K-T, IT98K-T, Gourgou-T and Nafi-T) and their respective non-transgenic near isogenic lines (IT97K, IT98K, Gourgou and Nafi) to Cowpea aphid-borne mosaic virus (CABMV) in greenhouse conditions. In a preliminary quality control test by enzyme-linked immunosorbent assay, the presence of Cry1Ab protein in transgenic seed lots ranged from 59% to 72%, with no significant differences among the lines (χ2 = 3.26;p = 0.35). Upon virus inoculation, all cowpea lines exhibited mosaic symptoms with similar severity between 7- and 11-day post-inoculation. No significant differences were observed in symptom severity. Significant differences were found between cowpea lines for time of symptom onset, virus accumulation in plants and days to 50% flowering. However, while comparing pairs of transgenic lines and corresponding non-transgenic lines, virus accumulation showed not significant differences whatever the pair. Time of symptom onset and days to 50% flowering did not also differ significantly between pairs of cowpea lines except Nafi/Nafi-T in which transgenic Nafi-T showed earlier symptoms (7.4 ± 0.7 vs. 8.9 ± 0.8 days post-inoculation) and shorter flowering time (37.3 ± 0.6 vs. 42 ± 1.7 days after sowing). Overall, these findings improve our understanding of the effects of Cry1Ab gene mediated genetic modification on cowpea infection by Cowpea aphid-borne mosaic virus, with potential implications for environmental safety assessment.展开更多
Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such v...Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such viruses is crucial for their detection and functional understanding of related genes.However,no studies have yet reported viral detection using antisera against movement proteins(MP)in these three viruses.In this study,we generated plasmids expressing three viral MP genes,and transformed them into the Escherichia coli strain,Rosetta,to recombinantly express and purify fusion proteins.Then,polyclonal antisera were derived by immunizing New Zealand white rabbits,after which western blotting was used to determine the titer,sensitivity and specificity of the antisera.The antisera titers against MP^(CABYV),MP^(MABYV) and MP^(SABYV) were 1:512000,1:256000 and 1:256000,respectively.The optimized working concentrations for the three antisera ranged between 1:10000 and 1:64000.Additionally,antisera against MP^(CABYV) and MP^(MABYV) only reacted with their corresponding MP proteins.Antiserum against MP^(SABYV) not only had the strongest reaction with its MP,but also reacted weakly with MP^(CABYV) and MP^(MABYV).All three antisera exerted no serological reactions with other poleroviruses.Furthermore,our data showed that all antisera specifically detected MPs in both Nicotiana benthamiana and cucumber leaves.Thus,we have established a system that sensitively detects three poleroviruses infecting cucurbits,using antisera against MPs.We provide a foundation for future research on the serological detection of these viruses,and interaction mechanisms between viruses and host plants.展开更多
文摘Cowpea (Vigna unguiculata L. [Walp.]) in one of the main grain legumes contributing to food security and poverty alleviation in Sub-Saharan Africa. To control the highly damaging legume pod borer Maruca vitrata F., transgenic cowpea lines expressing the insecticidal Cry1Ab Bt protein were developed. In this study, we evaluated the impact of Cry1Ab transgene expression on the susceptibility of four cowpea lines (named IT97K-T, IT98K-T, Gourgou-T and Nafi-T) and their respective non-transgenic near isogenic lines (IT97K, IT98K, Gourgou and Nafi) to Cowpea aphid-borne mosaic virus (CABMV) in greenhouse conditions. In a preliminary quality control test by enzyme-linked immunosorbent assay, the presence of Cry1Ab protein in transgenic seed lots ranged from 59% to 72%, with no significant differences among the lines (χ2 = 3.26;p = 0.35). Upon virus inoculation, all cowpea lines exhibited mosaic symptoms with similar severity between 7- and 11-day post-inoculation. No significant differences were observed in symptom severity. Significant differences were found between cowpea lines for time of symptom onset, virus accumulation in plants and days to 50% flowering. However, while comparing pairs of transgenic lines and corresponding non-transgenic lines, virus accumulation showed not significant differences whatever the pair. Time of symptom onset and days to 50% flowering did not also differ significantly between pairs of cowpea lines except Nafi/Nafi-T in which transgenic Nafi-T showed earlier symptoms (7.4 ± 0.7 vs. 8.9 ± 0.8 days post-inoculation) and shorter flowering time (37.3 ± 0.6 vs. 42 ± 1.7 days after sowing). Overall, these findings improve our understanding of the effects of Cry1Ab gene mediated genetic modification on cowpea infection by Cowpea aphid-borne mosaic virus, with potential implications for environmental safety assessment.
基金supported by the National Natural Science Foundation of China(31671995 and 31000840).
文摘Cucurbit aphid-borne yellows virus(CABYV),melon aphid-borne yellows virus(MABYV)and suakwa aphid-borne yellows virus(SABYV)are three poleroviruses that infect cucurbit crops.Developing specific antisera against such viruses is crucial for their detection and functional understanding of related genes.However,no studies have yet reported viral detection using antisera against movement proteins(MP)in these three viruses.In this study,we generated plasmids expressing three viral MP genes,and transformed them into the Escherichia coli strain,Rosetta,to recombinantly express and purify fusion proteins.Then,polyclonal antisera were derived by immunizing New Zealand white rabbits,after which western blotting was used to determine the titer,sensitivity and specificity of the antisera.The antisera titers against MP^(CABYV),MP^(MABYV) and MP^(SABYV) were 1:512000,1:256000 and 1:256000,respectively.The optimized working concentrations for the three antisera ranged between 1:10000 and 1:64000.Additionally,antisera against MP^(CABYV) and MP^(MABYV) only reacted with their corresponding MP proteins.Antiserum against MP^(SABYV) not only had the strongest reaction with its MP,but also reacted weakly with MP^(CABYV) and MP^(MABYV).All three antisera exerted no serological reactions with other poleroviruses.Furthermore,our data showed that all antisera specifically detected MPs in both Nicotiana benthamiana and cucumber leaves.Thus,we have established a system that sensitively detects three poleroviruses infecting cucurbits,using antisera against MPs.We provide a foundation for future research on the serological detection of these viruses,and interaction mechanisms between viruses and host plants.
