| The KIN1/PAR-1/MARK protein kinases are conserved in fungi and animals but not inplants. They are belonging to eukaryotic serine/threonine protein and involve in cell polarity,microtubule stability or cell cycle regulation. The homologs of KIN1/PAR-1/MARK familyin fungi was named KIN1. Saccharomyces cerevisiae has two homologs, named KIN1andKIN2. But all the other fungi we charactered in our study have only one KIN1homologs. Todate, KIN1kinase has not been studied in filamentous fungi and pathogen fungi. Wheat headblight or Scab was the second most important wheat disease after rust in China. Fusariumgraminearum is the most important pathegen fungus causing Scab. Rice blast is the mostimportant rice disease and its pathogen fungus Magnaporthe oryzae has been the modelorganism to study pathogen fungi.In this thesis, we generated the knock-out mutants F. graminearum KIN1gene viaSplit-PCR method, and the positive transformants were confirmed via PCR and southern blot.The research had confirmed that KIN1kinase was very important to the growth andpathogenesis of F. graminearum. We generated the KIN1-GFP fusion fragments under KIN1native promoters in the shuttle vector pFL2of E.coli, S. cerevisiae and F. graminearum. Thenwe randomly inserted the sequenced positive constructions to the kin1mutants viapolyethylene glycol (PEG) mediated transformation to get the complementary transformantsin F. graminearum. All of the positive complementary transformants were confirmed by PCRand KIN1-GFP fluorescent localization signal. The phenotypes of KIN1complementarystrains were recovered to the wide types in F. graminearum. The Fgkin1mutant had a50%reduction in pathogenecity in flowering wheat head, corn silks, and corn stem inoculations,but had no obviously changes in the synthesis of mycotoxin DON, which is used as animportant virulence factor in F. graminearum. The Fgkin1mutant reduced in conidiation.Most conidia of the Fgkin1mutant were shorter than that of the wide type PH-1. Most mutantconidia had just three septa which were less than four to seven in PH-1. The germination ofconidial middle compartments and hyphal branching were delayed in the Fgkin1mutant. Theseptation and nuclear distribution was uneven in the hyphae of the Fgkin1mutant. Energy was storaged as glycogen in animal and fungi. Glycogen accumulation ability was apparentlyreduced in ungerminated conidia but increased in ascus wall and mucilaginous fluid in theFgkin1mutant via glycogen stain in alive cells. The6dys old aerial mycelium on the potatodextroglucose agar medium was pushed down by sterilized0.1%Tween-20and then inducedunder black-light in constant temperature incubator at25degree centigrade to genenratesexual fruit bodies-perithecia. In the Fgkin1mutant, perithecia were still produced althoughthe number was reduced slightly compared to the wide type. The Fgkin1mutant peritheciastill fertilized but their sexual reproduction were defective. The ascus wall was dissolvingmuch earlier or aborted in the Fgkin1mutant. This indicated that KIN1involved in ascus walldevelopment. And this also can be confirmed by cell wall and hyperosmotic stress sensitivityin the Fgkin1mutant. These phenotype and the uneven septation indicated that FgKIN1maybe related to plasma membrane related cytokinesis. In the Fgkin1mutant, ascospore releasewas blocked and only small cirri was rarely observed on the top of perithicia in the Fgkin1mutant. Ascospores of the Fgkin1mutant germinated from one tip inside perithecia afterascus wall dissolved. These indicated that KIN1may involved in the ascospore germinationself inhibition, and this inhibition may related to normal energy metablism which wasregulated by KIN1. And this germination defective was also related to the culture inviroment.The dissolved ascus wall gave ascospores a different environment and culture condiationwhich was different to normal perithecia and complementary medium. FgKIN1localized tothe growth tip sides of septal pore areas where microtubule usually bundling. This localizationwas conserved in whole life cycles of both F. graminearum and M. oryzae. KIN1localizationwas not observed in the growth tip, dead cell septal pore area, closed septa pore area, andplasma membrane, etc. KIN1localized to functional septal pore area after septal formation.When septa emerging and growing, there was no KIN1localization signal boserved in septalpore areas. It is special to Fusarium that they have two β-tubulin genes while most fungi haveonly one. F. graminearum β-tubulin FgTUB1localized to nuclear in the Fgkin1mutant.Another F. graminearum β-tubulin FgTUB2localized to the microtubule cytoskeletons in theFgkin1mutant as that in the wide type PH-1. We introduced a point mutation in the kinasedomain of the FgKIN1gene sequence and then coloned this mutated sequence into vectopFL2to generate FgKINS172A-eGFP construction. The sequenced positive FgKINS172A-eGFPconstruction was inserted into the Fgkin1mutant genome via PEG mediated transformation togenerate FgKINS172A-eGFP kinase dead (inactivable kinase activaty) mutant. In the positiveFgKINS172A-eGFP kinase dead mutant, protein FgKINS172A-eGFP was still localized to septalpore but lost to recover most of the phenotypes except delayed ascus wall dissolving,observed ascospore shooting, cirrhi and ungermination after two weeks induction. The sexual reproduction and localization are kinase independent activities of FgKIN1while the otherphenotypes are kinase dependent activities. The septal pore localization may be controlled byKA1domain alone without any influence of kinase domain.In M. oryzae, the Mokin1mutant was also reduced in growth rate, conidiation, andpathogenecity. All of these reductions were similar to that in the Fgkin1mutant. KIN1-GFPalso localized to septal pore area in M. oryzae as in F. graminearum.Our studies in this thesis indicated that FgKIN1plays important roles in cytokinesis,asosopore release, ascopore germination, pathogenesis, and cellular energy metabolism, etc.FgKIN1kinase has both kinase dependent and kinase independent activities, and specificallyregulates FgTUB1. FgKIN1may be related to cell wall integrity development and plasmamembrane osmotic stress. KIN1has the similar functions in M. oryzae as that in F.graminearum. KIN1kinase localized to septal pore area in filamentous pathogen fungi, andthe localization, functions, and pathogenesis of KIN1kinase may be conserved in filamentouspathogen fungi. |
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