Functional Characterization of a Histone Lysine Demethylase Gene in the Rice Blast Fungus, Magnaporthe oryzae

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 이용환.The post-translational modification of histones plays important roles in regulating chromatin dynamics and transcription. It has been shown that disruption of proper modifications can lead to developmental defects and cancer in plants and mammals, respectively. Despite the generality of histone modifications as epigenetic mechanism in eukaryotes, implication of histone modifications in fungal pathogenesis is beginning to emerge. To date, two classes of histone demethylases have been identified: LSD and JmjC domain-containing family proteins. Here I reported identification and characterization of putative histone JmjC demethylases in the model plant pathogenic fungus, Magnaporthe oryzae. Combining BLAST and HMMER, seven genes encoding putative JmjC domain-containing histone demethylase were identified and named them as MoJMJ1 to MoJMJ7. Phylogenetic analysis showed that five of them belong to JARID, JMJD2, JHDM2 and JmjC-only domain groups, and the others were predicted to be fungal specific. Deletion of MoJMJ1, belonging to JARID group, resulted in defects in vegetative growth, asexual reproduction, autolysis, appressorium formation and invasive growth. The genetic approach was followed by biochemical approaches to examine the demethylase activity and identify target genes affected by steady state level of histone methylation. The expression of the genes, involved in signaling transduction pathways for appressorium development, and other TFs including MoHOX genes showed significant changes in ΔMojmj1. Introduction of native MoJMJ1 gene into ∆Mojmj1 restored not all but defects in vegetative growth, asexual reproduction, autolysis and appressorium formation, indicating the importance of regulating histone demethylation by MoJMJ1 during fungal development and appressorium formation. It is anticipated that this work would provide not only the insight into epigenetic regulation of fungal pathogenesis but also the knowledge that can be used in devising new control strategies against rice blast.ABSTRACT CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION MATERIALS AND METHODS I. Fungal strains and culture condition II. Sequence and phylogeneitc analysis III. Nucleic acid isolation and manipulation IV. Targeted deletion of MoJMJ1 mutants and complemented strains V. Developmental phenotype assays – Mycelial growth, conidiation, conidial germination, and appressorium formation VI. Pathogenicity test, wound inoculation, and sheath assay VII. Quantitative real-time PCR (qRT-PCR) VIII. Western blotting analysis RESULTS I. Identification of putative genes encoding JmjC histone demethylase in M. oryzae II. Domain architecture and conserved motifs in JmjC domain of MoJMJ III. Targeted gene disruption of MoJMJ1 IV. Vegetative growth of ΔMojmj1 mutants V. Asexual reproduction of ΔMojmj1 mutants VI. Conidial germination and appressorium development of ΔMojmj1 VII. Host penetration of ΔMojmj1 mutants VIII. Invasive growth of ΔMojmj1 mutants IX. Transcriptional regulation of the development and pathogenicity-related genes X. Immunoblot assay of MoJMJ1 XI. Complementation assay of MoJMJ1 DISCUSSION LITERATURE CITED ABSTRACT IN KOREANMaste