Functional Analysis Of Four Proteins Involved In DNA Methylation In Magnaporthe Oryzae

Heterochromatin protein 1(HP1),DNA methyltransferase 2(DIM2)and H3K9 methyltransferase(DIM5)play a major role on the heterochromatin formation,DNA methylation,histone modification,heterochromatic gene silencing and telomere maintenance.Rice blast disease,caused by Magnaporthe oryzae syn.Pyricularia oryzae,significantly reduces the yield of rice and threatens food security production.In order to elucidate the function of MoHP1,MoDIM2 and MoDIM5 in M.oryzae,we generated the deletion mutants of MoHP1,MoDIM2 and MoDIM5 by homologous recombination,analyzed the morphological characterization,pathogenesis,expression of telomerase reverse transcriptase(TERT)and telomere of wild type and deletion mutants.Comparing to wild type strain FJ 81278,MoHP1-52 deletion mutant shows reduced growth rate,significant less conidiophores,conidia reduction of 73%,no pathogenesis defect,increased dark pigmentation of colony,and significant upregulation of three melanin biosynthesis-related genes,ALB1,BUF1,RSY1.MoDIM5-23 deletion mutant shows reduced growth rate,but no conidiophores and conidia production defects and pathogenesis defect.MGG＿05052-105 deletion mutant shows significant less conidiophores,conidia reduction of 62%,no growth and pathogenesis defects.MoDIM2-102 deletion mutant shows no defect in growth,conidiophores and conidia production and pathogenesis.This demonstrated that MoHP1 involvs in regulation of vegetative growth,asexual production and secondary metabolism,MGG＿05052 involves in regulation of asexual production and MoDIM5 involvs in regulation of vegetative growth.MoHP1-GFP is localized in nuclear heterochromatin regions in the vegetative hyphae,conidia and invasive hyphae of wild type strain FJ81278.In contrast,MoHP1-GFP is diffuse throughout the nucleus with fewer foci in the MoDIM5 deletion mutant.Thus,the localization of MoHP1 depends on MoDIM5.The telomere patterns of wild type strain FJ81278 and seven deletion mutants were examined by southern hybridization.The telomeres of wild type strain FJ81278 that were detected with telomere repeat sequence(TTAGGG)is 11,and the telomeres of 7 deletion mutants are in the range of 9 to 14.The order of telomere number from small to large is ΔMoDIM5/ΔMoHP1-7、ΔMoHP1-52、ΔMGG＿05052-105、ΔMoDIM2-102、ΔMGG＿05052/ΔMoHP1-73、ΔMoDIM5-23、ΔMoDIM2/ΔMoHP1-40.The patterns of five shorter telomeric region of 7deletion mutant are similar to those of wild type strain FJ81278,but the patterns of six longer telomeric region are quite different from wild type strain.This demonstrated that MoHP1,MoDIM2,MoDIM5 and MGG＿05052 play an important role in the telomere maintenance in M.oryzae.Our results also suggested that the DNA methylation and histon modification of all detected telomeres may be different.The expression level of telomerase reverse transcriptase,TERT of wild type strain FJ81278 and HP1 deletion mutant was measured by q RTPCR.The expression of TERT significantly downregulates in HP1 deletion mutants comparing to the wile type strain FJ81278.The levels of TERT expression in HP1 deletion mutant were 43% of expression levels in wild type strain.Histone modifications in deletion mutants were determined by western blotting with specific antibodies.The levels of different kinds of histone lysine methylation were reduced or increased in different deletion mutants.Take together,these results can be helpful for elucidate the pathogenesis mechanism of M.oryzae from the perspectives of epigenetics.