Functions Of StpkaC1and StpkaC2in Regulating The Development And Pathogenicity Of Setosphaeria Turcica

Setosphaeria turcica causing Northern Corn Leaf Blight is an important fungal pathogenthreatening maize production. This disease often occurrs in the global low temperature andmoist hell corn producing areas. For a long time, the control of the disease still dependentson the resistant variety. But because the pathogen frequently mutates, often showesdifferent physiological differentiation and the toxins produced by different groups arevarious, it is very difficult to carry on the effective, accurate monitoring, resulting in therapid loss of resistance to maize varieties, to increase the difficulty of disease control.Earlier studies found that cAMP signal pathway played an important role in the regulationof nutrient mycelia growth and pathogenicity of S. turcica. Furthermore, a gene namedStpkaC1encoding the catalytic subunit of PKA was cloned and proved to be related withthe fungal growth, conidiation and invasion through RNAi technology. In order to illustratethe function of PKA regulating the growth and pathogenicity, StpkaC2, an isoform ofStpkaC1, was obtained in this experiment and researched through gene konck-outtechnology. The main results are as follows:1. Using bioinformatics methods, StpkaC2an isoform of StpkaC1, from S. turcicadatabase http://genome.jgi-psf.org/). StpkaC2owned an open reading frame of1209bpencoded402amino acid residues. The phylogenetic tree revealed that StpkaC2andStpkaC1, which both contained the conserved domains of PKA, belonged to differentclusters.2. The expression patterns detected with Real-time PCR showed that the expression levelof StpkaC2at3h past induction (hpi)(i.e. germ tube formation period) was2.2timesas high as that at0hpi (conidia), decreased slightly at6,12hpi (i.e. appressoriaformation stage and mature stage) and at24hpi (i.e. intrusive filament formation stage),it increased to4.2times as much as that at0hpi. Compared with StpkaC2, mRNA levelof StpkaC1at0,3,6and12hpi was similar and rose to3.2times as much as that at0hpi.3. Through protoplast transformation mediated by PEG, four strains of knockout mutantof StpkaC1and StpkaC2gene were identified with the hygromycin resistance screening,PCR and Southern blotting.4. Compared with wild type strain, the StpkaC1and StpkaC2gene knockout mutantsdisplayed faster growth rate, lightening colonial color, more aerial mycelia andsporulation defects.5. The content of intracellular melanin in mutants was lower than that of wild type strain. 6. The pathogenic penetration analysis showed that, on the artificial cellophane, StpkaC1knockout mutant hyphae formed appressoria, but appressorial formation was delayed,and the ability to penetrate on the artificial cellophane also reduced. While the StpkaC2knockout mutant was unable to form appressoria, but also could not penetrate theartificial cellophane.7. The StpkaC1and StpkaC2gene knockout mutant strains of crude toxin content weredetermined, and compared with the wild type the nockout mutant strains of crude toxincontent did not change significantly. But the pathogenicity of gene knockout mutants ofStpkaC1was significantly lower than that of the wild type, while gene knockoutmutants of StpkaC2fully lose the pathogenicity.