Functional Analysis Of StBCK1 Gene In CWI-MAPK Cascade Of Setosphaeria Turcica

Northern Leaf Blight of Corn caused by Setosphaeria turcica, is one of the most important diseases in corn production areas and often results in significant economic losses in epidemic years. Analyzing pathogenic regulatory mechanism and exploring more effective control measures have become a hot topic in the research field. A MAPKKK gene of S.turcica named StBCK1 was cloned in our previous research and target gene mutagenesis techniques was used to obtain StBCK1 gene deleted mutants. The founction of StBCK1 gene was identified by analyzing the characteristic of mutant. The main results were as follows:1. DNAstar, DNAMAN, and BLAST from NCBI website were used to analyze the gene structure of StBCK1. It was showed that StBCK1 gene had the conservative domains of MAPKKK-like gene in plant pathogenic fungi. Homology analysis showed that there was high homology between StBCK1 and BCK1genes of other filamentous fungi, suggesting that StBCK1 gene belonged to cell wall integrity pathway of S.turcica.2. StBCK1 gene homologous recombination plasmid were transformed into the protoplasts of S. turcica through PEG-mediated transformation. A StBCK1 gene disrupted mutant was obtained by screening of resistance to hygromycin B(Hgy+), and detecting by PCR and RT-PCR.3. The results showed that the function of StBCK1 gene was related to growth rate, mycelium and colony morphology, conidial production of S. turcica. Comparing with the wild-type isolate, the growth rate of StBCK1 gene disrupted mutant was obviously slower, and the colony colour was gray and became autolytic. Mycelium morphology of mutant was anomalistic, the mycelium color turned light, the hypha cell had no distinct septum and produced a lot of vacuole-like structures. The StBCK1 null mutant could produce lesser conidia than wild-type.4. The results revealed that StBCK1 gene could participate in the regulation of the cell wall development of S. turcica. Optical microscope observation displayed that the mycelia of StBCK1 gene null mutant were transparent, and scanning electron microscopy revealed that hyphae ofΔStBCK1 were coarse, porous in the surface. TheΔStBCK1 was hypersensitive to the cell wall-degrading enzyme treatment, producing protoplasts at a faster rate than wild type strain(WT),ΔStBCK1 releasing protoplasts as 20 times as WT. The sensitivity of StBCK1 null mutant was increasing with the content of the SDS or H2O2.5. The result revealed that StBCK1 gene also took part in regulating pathogenicity of S. turcica. The conidia of mutants could not penetrate the artificial cellophane, and could not infect the healthy and wounded corn leaves, suggesting that the StBCK1 gene null mutant lose its pathogenicity thoroughly.6. The results also revealed that StBCK1 gene could participate in the regulation of the melanin production of S. turcica.