MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica

The mitogen-activated protein kinase （MAPK）, a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK plays in phytopathogenic fungi. In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol （HOG） MAPK gene HOG1 of Saccharomy- ces cerevisiae. In addition, gene knockout technology was employed to investigate the function of STKI. Gene knockout mutants （KOs） were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain （WT）. Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface. STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum （HT）-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43. Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress. These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis. Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.

Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination,Appressorium Production,and Pathogenicity of Setosphaeria turcica

Systemic studies on the effects of mitogen-activated protein kinase （MAPK） signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126.

Stk2,a Mitogen-Activated Protein Kinase from Setosphaeria turcica,Specifically Complements the Functions of the Fus3 and Kss1 of Saccharomyces cerevisiae in Filamentation,Invasive Growth,and Mating Behavior

Setosphaeria turcica,an essential phytopathogenic fungus,is the primary cause of serious yield losses in corn; however,its pathogenic mechanism is poorly understood.We cloned STK2,a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum,56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae.To deduce Stk2 function in S.turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S.cerevisiae,a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S.cerevisiae.The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores,and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain.Moreover,STK2 complemented the function of KSS1 in filamentation and invasive growth,as well as the mating behavior of FUS3 in S.cerevisiae,however,its exact functions in S.turcica will be studied in the future research.

StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum

Homologous recombination(HR) and nonhomologous end joining(NHEJ) are considered the two main double-strand break(DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated St KU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in St Ku80. Phylogenetic analysis indicated that St Ku80 is most closely related to Ku80(XP001802136.1) from Parastagonospora nodorum, followed by Ku80(AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔSt KU80-1 and ΔSt KU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type(WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H2O2, but not to ultraviolet radiation. In summary, the St KU80 plays devious roles in regulating the development of E. turcicum.