Screening And Analysis Of Verticillium Dahliae-sensitive Mutants In Arabidopsis Thaliana

Verticillium wilt is a soil spread vascular systemic disease, which is caused by Verticillium dahliae kleb, distributting in the main cotton-growing region, and showing atrend of increasing spread. The pathogenic fungi has strong adaptability, a wide host range,an obvious pathogenicity differentiation, various ways of transmission and pathogenesis, complex mechanisms. Due to the lack of effective prevention and treatment drug,and lack of resistant materials to Cotton Verticillium wilt, making it more difficult to control this cotton disease. Breeding resistant varieties will be a feasible way to solve this problem by using modern molecular biological technology.When using Verticillium dahlia to infectArabidopsis,the cotyledons appear sallow, anthocyanin accumulation, plants were stunted and other typical symptoms of Verticillium wilt.The cotton has a large genome and a long growth cycle,slowing the progress to explore cotton resistant genes.The model plant Arabidopsis thaliana has great advantagesin the research of Cotton Verticillium wilt resistance mechanism because of itsadvantage, such as small genome,short growth cycle and so on.In this study,we breed the seeds of T-DNA socks on the 0.6% MS, vernalization for 3 days,thenput the good seedlings into soils after grown in 0.6% MS for 12 days,harvest the seeds per plant.These seeds were named with different numbers. The advantage of this method is even the mutant died, we can according to the number of seeds for next experiment. The seeds grown on 0.6% MS for 8 days, then placed them on the 1.0% MS. And theywere inoculated by placing a drop of fungal condia suspension(5x105 condia /mL) directly on the root tip.The seedlings appeared symptoms of Verticilliumwilt severely in soils.In this vitro system, we screened three mutunts: sr1, s63, s133.These provide materials for later research.Thesr1mutantgrown on 0.6%MS for 8days, inoculated with condia suspension,behaved more serious than WT after 8-12 days. The leaves faded green.Then they were almost chlorosis.The growth was inhibited after 20dpi(days post-inoculation). The result showed that sr1 mutantwas sensitive to V.dahliae.The pathogenic fungicontent of leaves was 8 times more than WT after 12 dpi. Thisexplained the reason of the sr1 mutant which sensitive to Verticillium wilt is caused by the number of colonies invades the body. Whensr1 mutant was transplanted into the soil for 22 days after 9 dpion theMS, the growth was inhibitedcompared to WT. The study found that the germination rateof sr1 was lower than WT. The root length of sr1 was shorter than WT significantly.The flowering time of sr1 was later than WT, but there was no difference between sr1 and WT after flowering.WT and sr1 grown in soil for 30 days, the freash weight of leaves and the root length were no significant difference between WT and sr1. The root length of mutant can resume to a certain extent when growing on MS medium in the absence of ammonium treatment. The study also found that the cell of elongation zone in the mutant is shorter than the WT. Genetic analysis indicated that the mutant is a single recessive gene mutation. The lowest reorganization rate was located in the front of chromosome V by map-based cloning.The s63 mutant grown in 0.6 MS for 8 days, and it was inoculated with condia suspension for 8 days.The leaves first faded green compared to WT, then were almost chlorosis.The number of chlorotic leaves increasedand the growth was inhibited.With time going on, its cotyledon began to dry up afterchlorosis,The degree of anthocyanin became serious with the increase of time. The life cycle was completed in advance companied with early bolting. The s63 mutant grown in 0.6MS for 20 days,and it was inculated following a root-tip procedure. Then they were grown in soil for 32 days. The number of chlorotic leaves of mutant was more than wide type. The relative content of anthocyanin in WT was 1.4 times that in s63 mutant after 18 dpi. The chlorophyll content in cotyledon declined2.5 times than the wild type.The pathogenic fungicontent of leaves was 7 times more than WT after 12 dpi. Trypan blue staining results showed that the dead cells in root tip or cotyledon were more than wild type.It,sshow that the s63 mutant is more sensitive to V.dahliae.The phenotype of the s133 mutant is similar to s63.After the s133 mutant was inoculated, the time of cotyledon became chlorosis was earlier than wild type. With time going on, its cotyledon began to dry up after chlorosis. The degree of anthocyanin became serious with the increase of time. The degree of senescence of mutant was serious than WT. Its life cycle was completed in advance companied with early bolting. The s133 mutant grown in 0.6% MS for 20 days, and it was inculated following a root-tip procedure. They were grown in soil for 32 days. The numberof chlorotic leaves of mutant was more than wide type. The relative content of anthocyanin in WT is 1.7 times that in s133 mutant after 18 dpi. The chlorophyll content in cotyledon declined1.5 times than the wild type.The result show that the s133 mutant is more sensitive to V.dahliae.