Functional Characterization And Identification Of STK1 Gene Encoding A MAP Kinase In Setosphaeria Turcica

Northern Corn Leaf Blight(NCLB),caused by Setosphaeria turcica which is a fungal plant pathogen,is a serious threat to maize production in North and Northeast China.It was reported that the growth,development and pathogenicity were regulated by cellular signal transduction in many fungi,MAP kinase signal transduction pathway,a universal pathway,have an important role in the ability of fungal plant pathogen to perceive changes in their environment and adjust their intracellular activities to regulate growth and development processes.STK1 gene isolated from S.turcica,encoded a mitogen-activated protein kinase(MAPK),which is homologous to Hogl in Saccharomyces cerevisiae and some other MAP kinases related to osmoregulation in other filamentous fungi.Our former study has found that it was related to conidial production and virulence of HT-toxin,but it was not clear the role of osmoregulation in S.turcica.We found it had exhibited striking sequence identity to MAP kinases related to osmoregulation in filamentous fungi.The functional complementation was used to confirm the function of the gene,which was performed through the expression of the vector containing STK1 gene in yeast HOG1 null mutant.Main results in this paper were as follows:1.We amplified the full length of the STK1 gene,which was an open reading frame containing 1071 nucleotides.The characterization of the gene by Bioinformatics showed that the ORF predicted 356 amino acids residues,which shared more than 90%homology to MAP kinases related to osmoregulation in other plant pathogenic fungi.Sequence comparison of predicted protein with other MAP kinases revealed the presence of all characteristic conserved subdomains,including the TGY motif which was the characteristic of hyperosmolarity-activated MAPKs and the site for threonine and tyeosine phosphorylation required for MAPK kinase activation,the CD domain and BD domain which were used to bind to activator,substrates and phosphatase.The aligned sequences were clustered primarily by the type of MAPK,forming three major clades: Hog1-homologue,Fus1/Kss1-homologue,and Slt2-homologue,and on this tree,Stk1 was classified with Hog1- homologous MAPKs subgroup.2.The yeast expression vector pVT102U-STK1 was constructed,and then it was transformed into the yeast HOG1 null mutant,and the positive transformants was selected by synthetic dropout medium and confirmed by PCR. 3.When expression of STK1 in budding yeast hog1â–³null mutant,the mutant phenotype was rescued by the complementation under salts stress,oxidative stress.This study exhibited that there was a similar signal transduction pathway in which the STK1 had a key function for resistance to environmental stresses.4.Most interesting,the STK1 showed better tolerance in 1M NaCl medium than wild type strains from the results of growth rate test and determination of intracellular glycerol. Because of its much more Na~+ than other elements in saline-alkali soil,we imagine a bright prospect in saline agriculture if STK1 was transformed and integrated in genome of crops.