Functional Study Of MAP Kinase Genes UeKpp2 And UeKpp6 In Ustilago Esculenta

Ustilago esculenta,a unique pathogenic fungus,could infect Zizania latifolia,to suppress host flowering and trigger host stem swollen called Jiaobai.It is a nutritial aquatic vegetable and medicinal material in East and Southeast Asia particularly in China.Previous studies had demonstrated that U.esculenta was a typical dimorphism fungus,similar to other dimorphism fungi that existed yest-like form and dikaryotic filament form,and the morphological transition of the two forms played pivotal role in their pathogenicity.In addition,mitogen-activated protein kinase(MAPK)pathways which widely existed in pathogenic fungi had significant functions on the dimorphism transition and pathogenicity,especially the MAPK genes,which were in-depth research at present.In this study,two MAPK genes UeKpp2 and UeKpp6 were cloned and identified from U.esculenta,based on fungal whole genome sequencing database and the amino acids sequences of Kpp2 and Kpp6 in Ustilago maydis.Also,the expression patterns,biological functions,of the two genes were studied by qRT-PCR,genetic modification,mating and innoculation assays.Meanwhile,the possible regulation pathway,which the two genes involved were explored by yeast two hybrid test.The MAPK gene UeKpp2 was cloned,and the sequence analysis showed that its open reading frame was 1065 bp,encoding 354 amino acids without introns,containing typical TEY conserved sequence of MAP kinases.Phylogenetic analysis showed it belonged to Fus3/Kss1 pathway,and had close relative to smut fungi,such as U.maydis,Ustilago hordei.Complementary assay indicated that UeKpp2 could complement the filamentous growth defect in U.maydis kpp2 mutant,and might promote the pathogenicity on a certain extent.One pair compatible haploid strains of? UeKpp2 were generated by protoplast transformation method based on homologous recombination.Further researches showed that ? UeKpp2 single mutant appear more multiple budding phenotype,but their growth rates were identical to the corresponding wild-type haploids.Moreover,the conjugation tube formation and mating ability were sharply attenuated compared with the wild-type strains.The exprssion profiling of UeKpp2 during growth and infecton progress indicated that it was continuously induced during hyphal growth and up-regulated at later stage during infection.In addition,the expression of UeKpp2 appeared difference both in T and MT type strains under different carbon and nitrogen sources stimuli,but was consistent to the hyphal growth ability.The results of yeast two-hybrid assays showed that UeKpp2 was able to interact with UeFuz7 and UePrf1.The expression of UeFuz7 was down-regulated during the later stage of filamentous growth and the expression of UePrf1 was up-regulated at 24 h in UeKpp2 mutant strains.Furthermore,?UeKpp2::UePrf1 were constructed and the mating ability could recover partly,indicating that UePrf1 acted on the downstream of UeKpp2 directly.UeKpp2 deletion influenced the pathogenicity,but it could form spores in Jiaobai according to the infection assay results.Another MAPK gene UeKpp6 was cloned and identified,and its open reading frame was 1737 bp,encoding 578 amino acids without introns.The protein contained serine/threonine dual specific protein kinase catalysis domain and had close relative to Kpp6 of U.maydis.UeT14?UeKpp6 and UeT55?UeKpp6 were obtained by PEG mediated protoplast transformation method.UeKpp6 mutant had no influence on morphology and growth status of haploid strains,and that conjugation tube growth and mating in vitro made no difference to wild-type strains.The expression pattern analysis of UeKpp6 showed that it was up-regulated during earlier infection stage and later filamentous growth phase in vitro,and the expression of UeKpp6 were different under different carbon and nitrogen sources stimuli.The results of yeast two-hybrid assays showed that UeKpp6 could interact with UePrf1,otherwise another four genes encoding proteins interacted with UeKpp6 were screened out from cDNA library of U.esculenta,including g1616,g3288,g6433,g2188.The expression of UeKpp2 was up-regulated more early and the expression of UePrf1 was down-regulated during later filamentous growth phase after UeKpp6 mutated.?UeKpp6 could infect Z.latifolia and form teliospores according to the infection assay.