Function Analysis Of STK2and StMSG5in Setosphaeria Turcica

A MAPK gene named STK2was obtained by searching genome database ofSetosphaeria turcica. The function of STK2was identified by heterologous genecomplementation technology. The result showed that STK2displayed similar functionswith Saccharomyces cerevisiae FUS3/KSS1in regulating filamentation, invasive growth,and ascospore development. A MAPK phosphatase gene named StMSG5was cloned andits function was preliminarily confirmed by gene knock-out technology. The resultsrevealed that StMSG5was involved in regulating penetration process. The mainly resultswere as follows:1. A MAPK gene STK2of S. turcica, which included1092bp cDNA sequence andencoded363amino acids, was cloned. The sequence analysis revealed that STK2sharedhighly homologous with other FUS3/KSS1-like genes of other plant pathogens.2. The yeast expression vector pVT102U-STK2was constructed. The reconstructedvector was transformed into the yeast fus3/kss1null mutant, and the positive transformantswas selected by synthetic dropout medium and confirmed by PCR and RT-PCR.3. The phenotype of fus3/kss1null mutant was rescued when STK2was expressed inbudding yeast fus3/kss1null mutant, suggesting that STK2shared the same functions inregulating filamentation, invasive growth and ascospore development of budding yeastcell.4. A MAPK phosphatase gene named StMSG5were found by searching the genomedatabase of S. turcica published on JGI website (http://genome.jgi-psf.org). The genecontained1521bp DNA sequence,1464bp coding region,2exons,1intron, and itencoded487amino acids. Sequence analysis revealed that StMSG5shared highlyhomologous with other MSG5-like genes of other plant pathogens and S. cerevisiae.5. Based on the basic plasmid pBS-pUC, a gene knock-out vector of StMSG5wasconstructed. The recombinated vector was transformed to the protoplasts of S. turcicamediated by PEG3350. A transformant were obtained by culturing on PDA mediumcontaining50μg/mL hygromycin B. At last, the mutant was determined through specialprmer PCR and RT-PCR.6. By compareing the characteristics of StMSG5gene knock-out mutant and thewild-type strain, the conidia output and appressorium penetration ability were increased, indicating that StMSG5was a negative regulatory factor in MAPK cascade. However,StMSG5gene knock-out mutant did not significantly change its mycelium morphology,colony characteristics, growth rate, suggestting that StMSG5did not regulate growth anddevelopment of mycelium.