Mechanism Of PKC Gene Regulating Pathogenicity Of Setosphaeria Turcica

Northern Corn Leaf Blight, caused by Setosphaeria turcica, is one of the important fungous diseases. The pathogenicity of S. turcica to maize is involved in cAMP,MAPK and Ca²⁺signal pathway. Protein kinase C (PKC), a serine/threonine kinases, is depended on phospholip and Ca²⁺. The protein kinase C gene of S. turcica, named as PKC, was cloned and analyzed in our Lab. After inoculating maize seedlings with the spore suspension containing PKC-specific inhibitor (chelerythrine), the spore could germinate and form normal appressorium on the leaf surface but invade maize seedlings from the stoma instead of penetrating directly leaf epidermis. There was no lesion appeared on the leaves three weeks after inoculation. The anti-sense vector was transformed into protoplasts of S. turcica by PEG-mediated transformation method and four PKC gene anti-sense mutants were obtained. Cultural characteristic, conidial germination, appressorium formation and pathogenicity of the mutants were studied in this paper to analyze function of protein kinase C. The results will be beneficial to understand how pathogenicity of S. turcica to maize is involved in cAMP,MAPK and Ca²⁺ signal pathway.1. The expression of PKC in S. turcica was increased on PDA media containing saccharose or NaNO₃ or Mg²⁺ ,the expression of PKC was decreased on PDA media containing Mn²⁺or Cu²⁺or Zn²⁺.The expression level of PKC was lower on PDA media containing sorbitol and the more concentration sorbitol, the less expression level. The expression of PKC was increased on PDA media containing NaCl(0.3⁰.5 mol/L), the expression of PKC was not changed on PDA media containing NaCl(0.7⁰.9 mol/L).2. We obtained four anti-sense mutants of S.turcica PKC gene which had been identified by hygromycin B resistance, PCR, semi-quantitative RT-PCR and Northern blot analysis. The growth rate of the mutants cultured on PDA media was same with wide type (WT), but mutants were much lower than WT on PDA media containing quinic acid (15 mmol/L). When conidia of WT and mutants were inoculated in 15 mmol/L quinic acid solution, the conidial germination ratio of WT was 90%, while that of the mutants were 3%⁵%. In contrast, culturing the germinated conidial in 15 mmol/L quinic acid solution, appressorium formed in WT but not in mutant strains. Penetration experiments showed that appressorium coming from WT could penetrate the cellophane but could not from mutants. Virulence of HT-toxin of mutants on maize leavies were the same with that of WT. While the inoculation of mutant strains did not cause visible lesions on the intact maize leaves, the inoculation of mutant strains caused visible lesions on the scratched maize leaves, but the lesion area was significantly reduced compared with WT.