Cloning And Functional Analysis Of MAPK Signal Transduction Genes In Setosphaeria Turcica

Mitogen-activated protein kinase (MAPK), also known as extracellular signal kinase (ERK), is a group of highly conserved protein kinases, which takes a great part in the diverse cell signal transduction pathways, such as osmotic regulation, cell wall integrate, morphologic formation, mating, virulence, growth and development. About 51 MAPK genes, involving in the growth, development, propaganda and pathogenicity of fungi, were cloned. Researches on the function of MAPK pathway in the plant fungal pathogen will not only benefit the understanding of the disease course, but also present some potent target genes for the disease control. Northern Corn Leaf Blight, whose causal agent is Setosphaeria turcica, is an important disease in corn leaves. To elucidate the function of MAPK pathway in the pathogen, the possible roles of the pathway in the hyphae growth, spore development, toxigenicity, and invasion were analyzed, 115 differentially expressed fragments which related to the target genes of the MAPK pathway regulation were acquired, and a gene, name as STK1 {Setosphaeria turcica mitogen activated protein kinase I) were cloned.rLuciferase/Luciferin(rL/L) system, a new method to detect the MAPK activity in fungus, was founded and proved to be effective and perspective in the study of MAPK pathway in microorganism. Both U0126 and TFP (inhibitor of CaM in Ca2+-CaM signal pathway) can concentration-dependently inhibited the MAPK activity in S. turcica, indicating that both of the two pathways participated in the MAPK activation in the fungal growth.The condium diluted with water or potato dextrose liquid medium could produce melanic appressoria in glass slide in 12 hours or in corn leaf surfaces in 24 hours. In a certain humid environment, temperature was the major factor to affect the appressorium production.There was no significant effect of U0126 on the filamentous growth and condial production of the 10 tested fungal plant pathogens: S. turcica, Curvularia lunata, Cochliobolus sativus, Fusarium oxysporum, Alternaria solani, Coniothyrium diplodiella, A. alternata, A. porri, Botrytis cinerea and A. brassicae, but the inhibitor can strongly inhibitthe spore germination of S. turcica, A. alternala, Cochliobolus sativus, Coniothyrium diplodiella and F. oxysporum, with no significant effect on the other tested pathogens. The effect was dose-dependant and will be decreased with the increase of treat time.U0126 and TFP could suppress the appressorium production of S. turcica in glass slide and corn leaf surfaces independently or mixed-treated. The effect was dose-dependant and would be disappear when 500 u M cAMP was added. The results indicated that the three signal pathways all involved in the regulation of appressorium formation of the pathogen.U0126 had significant effect on the production of the HT-toxin of S. lurcica. Some of the fractions in HT-toxin were inhibited and some new factions were induced. The inhibitor could concentration-dependently suppress the HT-toxin activity on corn leaves, but it was different between isolates.U0126 had no significant effect on the membrane leakage and PAL activity when suspended in distilled water with some ions in it, but the POD and PPO activity was significantly affected. The results may deduced that U0126 had no toxic activity to the cell integrate.115 cDNA fragments related to MAPK pathway regulation genes were acquired with optimized DDRT-PCR and reverse Northern blotting techniques.Sequencing of four differentially expressed fragments had shown that they were homogenous to genes related to TCA cycle, substance transmission in cell membrane, cell signal transduction, protein synthesis, reverse transcription, showing that MAPK pathway might take part in diverse life activities in the pathogen.The cDNA sequence, partial genomic sequence and the flanking sequences of a Setosphaeria turcica MAPK homogenous gene-STK1 gene were acquired by nest PCR and RACE. The gene included 3 introns (50bp, 52bp and 76bp respectively), some highly conserved domains, such as MAPK,...