Functional Analysis Of A NAD(H) Kinase Mopos5and Screening Of Its Interaction Proteins In Magnaporthe Oryzae

Rice blast disease, caused by Magnaporthe oryzae, is one of the most destructive and prevalent diseases in global rice production, which severely restricts the food security worldwide. Although in the past decades of years, systematic research on the growth, development and pathogenicity of this fungus has been carrying out effectively, the mechanisms of colonization and expansion are still not well-understood. Host-driven ROS seem to be the first major barrier for the colonization and expansion of pathogens, while NAD(H) kinases play an important role in ROS scavenging system through regulating the fine intracellular balance between NAD(P) and NADP(H). Therefore, functional analysis of a NAD(H) kinase MoPos5in M. oryzae and exploration of the signal pathways which it is involved in will be conductive to further understanding of the molecular mechanisms of colonization and expansion of this fungus in rice plants.MoPos5, containing typical conserved motifs and domains of NAD(H) kinase, can functionally complement the growth deficiency of the POS5deletion mutant of Saccharomyces cerevisiae under oxidative stress. During the whole life and infection cycle of M. oryzae, MoPos5always kept in a relatively stable transcription level, and its subcellular localization was roughly considered to be mitochondria with the assistance of Janus Green B staining.MoPos5was proved to be closely related to the mycelia growth, conidiation and pathogenicity of M. oryzae. The deletion mutants showed obviously mycelia growth deficiency compared to the wild type strain. Due to the downregulation of MoNoxl in deletion mutants, which is involved in intracellular ROS generation, it was indicated that disfunction of MoPos5might cause negative effect on intracellular ROS scavenging systems, which then brought about cytotoxicity and the final growth defects. The conidiations of deletion mutants were also deficient, and it had been demonstrated that the decreased production of conidiophores was the primary cause. However, the conidia produced by the deletion mutants possessed normal morphology, and they could also germinate to form effective appressoria just like the wild type strain. Nevertheless, after penetration into host tissues, the mutants could not fulfill colonization and expansion successfully, which were proved by the microscopic observation and biochemical staining for the ROS accumulation around the infection site, so the pathogenicity of mutants fatally reduced. Besides, MoPos5showed to be essential for utilizing linolenic acid as carbon source, which might be connected with its NADPH generation that was necessary for β-oxidation of linolenic acid. Moreover, overexpression of MoPos5had nothing to do with fungal growth, development and pathogenicity, except for slight increase of conidiation and upregulation of MoNoxl transcription.Other two NAD(H) kinase genes MoNADK2and MoNADK3were both upregulated in MoPos5deletion mutants, which indicated that there was a certain degree of functional redundancy among those three NAD(H) kinases in M. oryzae, and MoNADK2and MoNADK3might partially make up for the deficiencies caused by MoPos5deletion, such as supplying an essential amount of NADPH for survival.Drawing support from yeast two-hybrid system, nine MoPos5interaction proteins were obtained, six of them were gained from screening the mycelia cDNA library of Guy11, and the other three were the homologous of Pos5p interactions proteins in M. oryzae. According to bioinformatical predictions and relevant research papers, the MoPos5interaction proteins were likely to participate in various biological processes, including cytoskeleton morphogenesis, cell movement, cell division, cell ageing and death, maintainance of mitochondrial function, defense response to environmental stress, material and energy metabolism and signal transduction.In conclusion, it appeared more likely that NAD(H) kinase MoPos5regulated both intracellular and extracellular ROS levels through diverse effectors, and was thereby directly related to the growth, conidiation, colonization and expansion in host tissues of rice blast fungus.