The Functional Analysis Of StMSN2 Gene In Setosphaeria Turcica

In our previous study,tolerance to hyperosmotic stress of StHOG1 gene knock-out mutants in the HOG-MAPK cascade pathway of Setosphaeria turcica is reduced,which partly is attributable to the decreased ability of mutants mycelia to accumulate glycerol.The tolerance of StHOG1 gene knock-out mutants to phenylpyrrole fungicides as well as cell wall synthesis interferents is greatly enhanced.Electron microscopic observation revealed that the floccule outside the mutants cell wall disappeared and the cell wall becomes smooth.We hypothesize that mutations in HOG1 gene also affect the development of mycelial cell walls.However it is unclear whether this change in cell wall is related to the increased tolerance of pathogens to phenylpyrrole fungicides and cell wall synthesis interfering substances.Studies have confirmed that HOG1 can phosphorylate transcription factors such as SKO1,HOT1,SMP1,MSN1,MSN2 and MSN4,but how these transcription factors regulate the expression of target genes is not clear.In our study,we found that there is only one homolog of Msn2/Msn4 in S.turcica.Studies have shown that the tolerance of double mutants of the yeast MSN2 and MSN4 to hyperosmotic stress is greatly reduced.MSN2 Knockout Mutants of Magnaporthe grisea no longer produce conidia and lose the ability to infect intact rice leaves.This shows that Msn2 protein is likely involved in the adaptation of the fungal HOG-MAPK cascade pathway to hyperosmotic stress and the regulation of cell wall development and pathogenicity.However,it is not clear whether the Msn2 protein is involved in the regulation of pathogen resistance to phenylpyrrole fungicides and to cell wall synthesis interfering substances.On this basis,the StMSN2 knockout mutants were obtained by Agrobacterium-mediated transformation technology and the growing and developmentting characteristics,pathogenicity to maize,tolerance to hyperosmotic stress,tolerance to phenylpyrrole fungicides as well as cell wall synthesis interferents of StMSN2 knockout mutants were studied.The main results were as follows:1.StMSN2 gene knock-out mutants were obtained in the study.StMSN2 gene knockout recombinant plasmid was constructed by means of pBS-PUC and pPZP100 vector.Based on the principle of homologous recombination,two mutants,which named ΔStMSN2-1 and ΔStMSN2-2 with resistance to hygromycin B,were obtained by Agrobacterium-mediated transformation technology.These mutants were further determined through specific-primer PCR,RT-PCR and Southern blotting.2.StMSN2 gene knock-out affect the growth and pathogenicity to maize of pathogens.Compared with WT,the mutants’ colony growth rate were increased.The mutants conlony appeared white and the mutants did not produce conidia.Optical microscope observation indicated that the hyphae cells of the mutants were longer than those of WT and mutants cells length is approximately 3.2 times that of wild-type cells.Melanin content in the mutants were also decreased.Deletion of the StMSN2 gene greatly delays the differentiation into appressorium of mutant mycelium.Hyphae penetration test showed that the hyphae of WT could penetrate the cellophane,while the mutants failed.StMSN2 knockout mutants failed to infect intact maize leaves,but could penetrate and colonize in the stabbed maize leaves.3.The deletion of StMSN2 gene enhanced the tolerance of the pathogen to cell wall synthesis interfering substances.And it reduces the resistance of cell wall to cell wall lytic enzymes.(1)The colony growth inhibition ratio of the mutants were significantly lower than that of WT under CFW and Congo red treatment.(2)The protoplasts produced by dissolving StMSN2 gene knockout mutants hyphae using cell wall degrading enzymes were 1.56 times more than those produced by dissolving WT hyphae.4.The deletion of StMSN2 gene reduced the tolerance of pathogens to hyperosmotic stress.Hyperosmotic stress significantly inhibited the growth of mutants colonies than wild-type.The colony growth inhibition rates to ΔStMSN2-1,ΔStMSN2-2 and wild-type strains respectively were 59.6%,60.6% and 11.6% under 0.6 M sorbitol treatment.The colony growth inhibition rates respectively were 49.2%,57.5%,and 30.1% under 0.4 M NaCl medium treatment.The colony growth inhibition rates of 0.4 M KCl respectively were 51.2%,53.0%,and 40.1% under 0.4 M KCl treatment.5.The StMSN2 knockout mutant showed no significant change in the tolerance to fludioxonil and iprodione.However,the StHOG1 knockout mutants shows significant increase in tolerance to fludioxonil and isofluril.Under the 35 μg/mL iprodione treatment,the colony growth inhibition rates of WT strain,StMSN2 knockout mutant and StHOG1 knockout mutant respectively were 100%,100%,100% and 7%.Under the 0.75 μg/mL malononitrile treatment,the colony growth inhibition rates of the WT strain,the two StMSN2 knockout mutants and the StHOG1 knockout mutant respectively were 80%,82%,77% and 9%.