Analysis Of Physiology And Genetics To Verticillium Dahliae-Related Mutants In Arabidopsis Thaliana

Verticillium wilt greatly restricts the production and development of cotton and related industries.When cotton infected by Verticillium wilt,the macula appears in the edges or main veins of leaves and become big and withering,the leaves dry up and cotton boll shed finally.It affects the yield of cotton and the length and intensity of cotton fiber seriously.Due to complex genetic background and long growth cycle for cotton,which makes it difficult to prevent Verticillium wilt.Arabidopsis thaliana is a model plant,it is the host of Verticillium dahliae,and it has clear genetic background,short growth cycles,small individuals and homozygous genes,it can be used as a great study material for the exploration controlling methods of Verticillium dahliae and resistant gene of cotton.Our group have constructed a screening method for Verticillium dahliae-related mutants in Arabidopsis thaliana in the early stage.First,phenotypic validation of these mutants in Arabidopsis thaliana was carried out.And the mutant se739 and se105-6 was obtained by verifying the phenotype,which is sensitive to Verticillium dahliae.On this basis,we preliminarily analyzed the characteristics of resistance of these two mutants to Verticillium dahliae.Firstly,we analyzed the disease-resistant characteristics of the se739.The seedlings of the se739 on solid medium were inoculated with Verticillium dahliae for further culture.Compared with Col-0,with the increase of cultured processing time,the se739 has more obvious symptoms of disease,leaves faded green and turned yellow,the accumulation of anthocyanin and the chlorophyll content of infected leaves reduced significantly.The result of trypan blue staining showed that the number of dead cells in the leaves of se739 was more than that of Col-0 upon infection with Verticillium wilt.The extracts from infected leaves were cultured on PDA culture medium,and the results showed that the pathogenic fungi content of se739 on infected leaves was more than that of Col-0.The content of Verticillium dahliae in Arabidopsis thaliana was detected by qRT-PCR,the results showed that the content of Verticillium dahliae in the se739 nearly was 3.7 times as much as the Col-0.The results of these experiments indicating that the se739 on solid medium is susceptible for Verticillium wilt.In order to analyze the disease-resistant characteristics of the se739 in the soil,four-week-old the seedlings of the se739 and Col-0 in the soil were inoculated with Verticillium dahliae for further culture.With the increase of cultured processing time,the se739 has more obvious symptoms of disease,including more severe stunting,wilting,anthocyanin accumulation,decline of chlorophyll,tissue necrosis,and the disease index of plant increasing,when compared with Col-0 plants at 25 day post inoculation.The results of these experiments indicating that the se739 mutant is susceptible for Verticillium wilt.Genetic analysis indicated that the se739 mutant was a single recessive gene mutation.Through mapbased cloning,we know that the mutated gene of the se739 is located in the marker K18P6.We know that the se739 is a mutant about the TTG gene.Gene sequencing shows that the 681 th base guanine was replaced by adenine,this trigger the change of WD40 protein sequence.Then,we analyzed the disease-resistant characteristics of the se105-6.The seedlings of the se105-6 on solid medium were inoculated with Verticillium dahliae for further culture.Compared with Col-0,with the increase of cultured processing time,the se105-6 has more obvious symptoms of disease,leaves faded green and turned yellow,the accumulation of anthocyanin and the chlorophyll content of infected leaves reduced significantly.The result of trypan blue staining showed that the number of dead cells in the leaves of se105-6 was more than that of Col-0 upon infection with Verticillium wilt.The content of Verticillium dahliae in Arabidopsis thaliana was detected by qRT-PCR,the results showed that the content of Verticillium dahliae in the se105-6 nearly was 40 times as much as the Col-0.The results of these experiments indicating that the se105-6 on solid medium is susceptible for Verticillium wilt.In order to analyze the disease-resistant characteristics of the se105-6 in the soil,four-week-old the seedlings of the se105-6 and Col-0 in the soil were inoculated with Verticillium dahliae for further culture.With the increase of cultured processing time,the se105-6 has more obvious symptoms of disease,including more severe stunting,wilting,anthocyanin accumulation,decline of chlorophyll,tissue necrosis,and the disease index of plant increasing,when compared with Col-0 plants at 25 day post inoculation.The results of these experiments indicating that the se105-6 mutant is susceptible for Verticillium wilt.Genetic analysis indicated that the se105-6 mutant was a single recessive gene mutation.Through mapbased cloning,we firstly know that the gene of the se105-6 mutant was located in the marker MQB2.