Functional Analysis Of Transcriptional Factor MOAP1-Regulated Genes In Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast, caused by Magnaporthe oryzae, is a destructive causal agent observed during rice cultivation that severely threatens the production of rice crops. Recently, Control measures such as the use of disease-resistant rice cultivars and application of fungicides have not completely eradicated the disease since newly developed disease-resistant rice cultivars were prone to lose resistance after emergence of a new pathogenic or anti-fungicide population. Thus, the control of rice blast disease remains a serious problem, and the characterization of fungal development and pathogenicity-related molecular mechanisms is crucial to prevent the disease. Currently, the availability of the genome sequences of both rice and the fungal pathogen have not only accelerated the discovery of pathogenesis related genes but provided a new platform to understand molecular pathogenesis at the genome level. In this paper, we detailed elucidated the function of three genes regulated by the transcriptional factor MoAp1 during development and pathogenicity of M. oryzae, and the main results are showed as follows:First, Gti1/Pac2 family protein played important roles in development and pathogenicity of M. oryzae.Previous study indicated MoAp1 is a positive transcription factor that regulates the transcription of a series of genes important in growth, development, and pathogenicity of M. oryzae. To identify target genes of MoAp1, we screened the â–³Moap1 mutant SAGE database and identified MoGTI1 （MGG₀8850） and MoPAC1 （MGG₀6564） as paralogs to Gti1 and Pac2 of S. pombe and other fungi. Gti1 and Pac2 are conserved family proteins that regulate morphogenesis in fungi. Here, we found that MoGti1 and MoPac2 contained the typical Gti1/Pac2 family domain and had distinct roles in vegetative growth and stress responses. Deletion of MoGTI1 resulted in more sensitive to hydrogen peroxide, iron and osmotic stresses, lost ability for sexual reproduction, reduced conidian and produced abnormal conidia, reduced appressorial formation and failed to develop invasive hyphae in planta. Whereas the deletion of MoPAC2 lead to conidial production was obvious increased and the deletion mutant reduced pathogenicity in the AMopac2 mutant corresponded with an increased expression of several plant defense genes, including PR1a, AOS2, LOX1, PAD4, and CHT1.Second, identification on Gtil/Pac2 family protein-regulated proteins of M. oryzae and site mutantion of MoGtil.To further understand the regulation mechanism of MoGtil and MoPac2, we used isobaric tags for a relative and absolute quantitation （iTRAQ） assay to analyze different of the mutants’proteome compared with wild-type and found that expression of a series of proteins was changed in â–³Mogtil and â–³Mopac2 mutants, including three pathogenic-related proteins MoMpgl, MoRacl and MoCmkl. Especially, MoGtil and MoPac2 through regulate the expression of the hydrophobin pathogenicity factor MoMpgl to control surface hydrophobicity, and constructive expression of MoMPGl in the AMogtil and AMopac2 mutants were only restored defects in surface hydrophobicity but not pathogenicity, which suggested that there were other pathogenic factors functioned downstream of MoGtil and MoPac2. Bioinformatics analysis showed that MoGtil contain several functional site, To further elucidate the function of MoGtil, domain deletion and site mutants were performed, we found that nuclear localization signal （NLS）, a putative protein kinase A （PKA） phosphorylation site （69 aa）, and a potential leucine-rich nuclear export signal （NES） were necessary for the function of MoGtil, and we also found that the expression and localization of MoGtil were regulated by protein kinease MoPmkl. Our studies provide a comprehensive analysis of two Gitl/Pac2 family proteins in a plant pathogenic microorganism and underline the conserved important functions of Gti/Pac2 family proteins.Third, flavodoxins-like protein MoYcp4 played important roles in development and pathogenicity of M. oryzae.Chapter â…¢ of this study, we characterized the functions of MoYcp4, the homology of Saccharomyces cerevisiae ScYcp4, functioned downstream of MoApl and was a potential target protein of MoApl. Transcriptional levels analysis showed that MoYCP4 was obvious up-regulation during conidiation, appressorium and infection stages. Further gene knocked out and found the growth rate of â–³Scycp4 mutant was slight reduced, but the conidial production was obvious increased, over 10 fold compared with wild-type. Although the rate of appressorial formation was not affected, the appressorial turgor was distinct reduced, the ability of infection on rice and barely was reduced, finally lead to reducing pathogenicity. In summary, MoYcp4, a target protein of MoApl, involved in growth, conidiogenesis, infection and pathogenicity of M. oryzae. Our studies provide a comprehensive analysis of flavodoxins-like protein and promote the study about pathogenic-related molecular mechanism in M. oryzae.Collectively, our study indicated that MoGtil, MoPac2 and MoYcp4, which were regulated by transcriptional factor MoApl, played important roles in growth, development, conidiogenesis and pathogenicity of M. oryzae. Our results facilitated to deep elucidate the mechanism of gene network regulated by MoApl during growth, development and pathogenicity of M. oryzae, and also provided a very helpful reference value for developing effective fungicide pointed at as the gene regulation network for target.