| Rice false smut is a rice disease caused by pathogenic fungus Ustilaginoidea virens. It has been reported world-widely and become a major disease in rice production in China. The pathogen does not only cause yield loss, but also produce ustiloxins which are harmful to human and live-stocks. However, U. virens is under-investigated. It is still controversial about its life cycle, infection type and so on. Gene functional analysis for this pathogen mainly relies on gene expression profiling and screening among random insertional mutants libraries. Due to no report on gene knock-out approach yet, studies on gene functional analysis for U. virens are severely limited.In this study, we analyzed and compared different methods for fungal transformation and tried to set up gene knock-out approach by Agrobacterium tumefaciens mediated transformation or PEG mediated protoplast transformation. We successfully constructed this approach based on ATMT. The study firstly re-constucted binary vector p CAMBIA1300 into gene knock out vector p CBDW for U. virens and functional complement vector p CBDW-GEN. And then vectors for Uv HOG1 gene knock out and complement were constructed. With modified ATMT system, we set up a gene knock out system for U. virens and generated Uvhog1 mutant among 617 transfomants. With the same approach, we further knocked out 5 NRPS genes to test the homologous recombination efficiency of the system. The result showed the general efficiency of U. virens gene knock out is 1.67%, which is acceptable for more genes knock out. During gene functional analysis for Uvhog1 mutant, the defect strains showed slower growth, reduced branch angle, and defect in pigment synthesis; althouth the morphology of conidia and germling were not affected, conidiation was significantly reduced. The defect of Uv HOG1 not only increased the sensitivity of the mutant to hypersomosis, but also defected osmotic regulation by accumulating sorbitol and glycerol, and at the same time, Uv ATF1 and Uv SKN7 were expression abnormally when Uv HOG1 defected. Besids, Uvhog1 mutants showed higher sensitivity to protoplast stress and irrelevant to cell wall stress. During the growth of rice seedlings, culture filtrates of Uvhog1 mutant showed reduced inhibition effect.Compared with rice false smut, Fusarium head blight is a disease on wheat maily caused by Fusarium graminearum.This disease is becoming severe world widely, causing yield loss and food security issues. To better characterize the HOG1 gene in different pathogenic fungi, the study knocked out Fg SSK2, Fg PBS2 and Fg HOG1 in Fg HOG1 pathway. The knock out of any element in this pathway caused reduced growth rate and conidiation; mutants were not only sensitive to hyperosmosis, but also lost the ability to adjust osmosis by accumulating glycerol, mannitol, arabilol and sucrose; besides, all the mutants were sensitive to oxidative stress and protoplast stress, except cell wall stress. The mutants of Fg HOG1 pathway were also defect in female fertility, DON synthesis and pathogenicity.The study constructed the first gene knock out approach for U. virens and successfully generated Uvhog1 mutant, which is a strong support for U. virens functional genome analysis. Also, by analyzing and comparing the functions of HOG1 in F. graminearum and U.virens, it was better understood in its conservation of vegetative growth, conidiation, stress responses, cell integrity, sextual reproduction, pathogenicity and virulence and also provide hypothetical support on disease management for rice false smut and Fusarium head blight. |
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