Cloning And Functional Analysis Of The Type 2A Phosphoprotein Phosphatase Gene In Setosphaeria Turcia

Corn northern leaf blight, caused by Setosphaeria turcica, is an important disease in maize production and often causes serious economic losses. Studies have shown that growth and development of many plant pathogenic fungi is subject to cell transmembrane signal transduction, such as the MAPK signaling pathway, cAMP pathway and Ca2+ signaling pathways, etc. Research on the regulation mechanisms of signal transduction pathways involved in the growth and development of corn northern leaf pathogen, will help us understand the molecular mechanism of the development and infection of phytopathogens, and lay theoretical foundation for the study about the interaction mechanisms between host plants and pathogens and plant disease control.Type 2A phosphoprotein phosphatase (PP2A) is a major Ser/Thr phosphatase. PP2As have been shown to play roles in the regulation of the cell cycle, signal transduction and metabolism by controlling gene transcription, RNA splicing, and translation in a variety of organisms.In order to study the relationship between PP2A and conidia germination, appressorium formation and pathogenicity in S. turcica, cantharidin, the specific inhibitors of PP2A, was used to study the function of PP2A.Cantharidin showed significant inhibition effects on hyphal growth and conidial germination. The higher was the inhibitor concentration, the lower was the germination ratio of spores. Cantharidin caused the germ tube shortening and cell swelling irregularly. In addition, cantharidin could also obviously decrease the biological activity of HT-toxin. It was indicated that PP2A might regulate hyphal and conidial germination by controlling the cell polarity, and also involved in the biosynthesis of HT-toxin in Setosphaeria turcica.Degenerate oligonucleotide primers were designed based upon conserved nucleotide sequences of the known PP2A-C gene andPP2A-B gene in plant pathogenic fungi. We amplified their homologous fragments from S. turcica 01-23 genomic DNA. Then its flanking regions were obtained by Genome Walking. We got a PP2A-C full length DNA sequence of 1600 bp and a DNA homologous fragment of PP2A-B which is 2425 bp in length. The specific probe of PP2A-C was prepared to apply for Southern blotting. The results showed that PP2A-C gene gene only had single copy in genomic DNA of S. turcica.To study the gene function deeply, the vector of homologous recombination of PP2A-C gene was constructed, and the protoplasts of S. turcica were transformed with it through PEG-mediated transformation system. 15 transformants were obtained after screening with hygromycin B. It was determined through Southern blotting that the transformant E was mutant. In contrast to the wild strain 01-23, the PP2A-C mutant showed different phenotypes at the same growth conditions. On PDA medium, filamentous growth of PP2A-C mutants was impaired. Pathogenicity assays showed that the pathogenicity of the mutants was decreased.These results indicated that PP2A gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, which could lay a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.