Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this stu...Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.展开更多
Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana indust...Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia(66.7 mg/ml) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.展开更多
基金supported by the National Natural Science Foundation of China (30860149 and 31360364)the Joint Support Program from Tropical Crop Breeding Engineering Center of Ministry of Education of China+1 种基金the Crop Science National Key Disciplines of China (lhxm-2012-2)the Key Scientific Research Program from Hainan Province,China (ZDZX2013023)
文摘Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.
基金Supported by Yunnan Science and Technology Innovation Talent Program(2015HA034)National Natural Science Foundation of China(NSFC31560505)
文摘Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4(Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia(66.7 mg/ml) of wild-type strain of Foc TR4(WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplast formation, which consisted of 25 mg/ml driselase, 0.4 mg/ml chitinase, 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×10~7 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per microgram of DNA were obtained. AmCyan, RFP and YFP genes were stably transferred into the WT-Foc TR4, separately, using the protoplast transformation system. The gene FoOCH1 encoding α-1, 6-mannosyltransferase in the WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plant-pathogen interactions.
