The Mechanism Of Catalytic Subunit Of CAMP-dependent Protein Kinase A Regulating Pathogenicity Of Setosphaeria Turcica

Setosphaeria turcica, an important fungal pathogen, is the threat of corn production. Lots of researches found that cAMP signal transduction pathway could regulate the growth, development and pathogenesis of pathogenic fungi. The components of cAMP pathway include protein kinase A and adenylyl cyclase, and protein kinase A is the main effector of cAMP signal transduction pathway, which is directly related to the pathogenecity of pathogenic fungi.RACE technology was used to obtain the full length sequence of cAMP dependence protein kinases A catalytic subunit gene (PKA-c). The bioinformatic analysis revealed that the deduced amino acid sequence of the PKA-c owned 97% identity with Pyrenophora tritici-repentis (XP₀01939935.1) and 92% identity with Alternaria alternate (ABY83137.1).The cDNA and DNA sequences of PKA-c were compared by DNAMAN, the ORF (open reading frame), which was interrupted by two introns, was 1524 bp long, and encoded a protein with 507 amino acid residues. The expression of this gene was analyzed on mRNA level; we found PKA-c involving in the utilization for carbon source, influenced by the osmotic stress from sorbitol, and participate in stress reaction of plant pathogenic fungi under hypertonic environment by sodium salt. This research laid a foundation for the functional analysis about cAMP signal transduction pathway in phytopathogenic fungi.RNAi (RNA interference) technology has been widely applied in the study of gene function due to its efficiency and economy. To further define the relationship between PKA-c gene and pathogenesis of S. turcica, we used RNAi (knockdown) as a tool for targeting endogenous gene PKA-c in S.turcica. PEG mediated transformation of PKA-c RNAi plasmid into protoplasts of S.turcica. The transformants were screened by hygromycin B and PCR (polymerase chain reaction) with specific primers corresponding to hygromycin. Three mutants M3, M5, M9 were obtained by semi-quantitative RT-PCR and Nouthern bloting analysis performed. Phenotypic analysis of these mutants showed that the extend of mutant hyphae grew faster than the wild isolate, conidia production was reduced, germination required longer periods of time and their pathogenicity significantly reduced, but crude HT-toxin activity between mutant and wild type were not strikingly difference. The decreased expression of PKA-c gene accompanied with melanin transcription factor, xylanase gene and tubulin gene expression decrease, while the xylanase of wild strain and mutant strain were extracted, enzyme activity was measured and the result showed that mutant had low activity. Therefore, It was concluded that PKA-c gene was involved in regulating conidia production and germination of S. turcica, and related to synthesis of melanin and activity of xylanase not directly relate to the activity of HT-toxin.