Function Analysis Of 12 Peroxisome Biosynthesis Genes In Magnaporthe Oryzae

Peroxisomes, single-membrane bounded organelles, are involved in various metabolisms in eukaryotic cells. Peroxisomes originate from the endoplasmic reticulum. Since peroxisomes do not contain their own DNA, the matrix enzymes and membrane proteins are encoded by nuclear genes, synthesized in the cytoplasm, and then recognized and transported to the organelles relying on their peroxisomal targeting signal (Peroxisome targeting signal, PTS). Proteins involved in peroxisome biogenesis are designated as peroxins and encoded by PEX genes.In recent years, peroxisomes function and the formation were paid more and more attention in filamentous fungi. With the advance of functional genomics and transformation techniques, more than 30 PEX genes have identified in filamentous fungi. The complicated interactions were found in different PEXs in peroxisomal biogenesis. Functional divergence also presented in homologues of same PEX in different species. However, the peroxisomal formation and degradation associated genes are limited reported in filamentous fungi, especially in plant pathogenic fungi. Magnaporthe oryzae has typical infection process, pathogenesis and genetic characteristics in plant pathogenic fungi. In present work, we therefore investigated systematically the genes related to peroxisome biogenesis in M. oryzae.We first refined the homologues in M. oryzae of PEX and ATG genes using bio-informatics strategies. Twenty-eight PEX genes, with the exception ofPEX15, PEX17, PEX18, PEX21 and PEX22, were detected in M. oryzae genome, including the PTS receptors PEX5 and PEX7 which recognizes PTS1 and PTS2 respectively, the ring complex proteins MoPEX2, MoPEX10, MoPEX12. Some filamentous fungi-specific genes were also found such as MoPEX16, MoPEX14/17 and MoPEX20, indicating specificity of the peroxisome biogenesis in fungi. These proteins were usually conservative in M. oryzae, yeast and F. graminearum, especially among filamentous fungi. All the homologous genes between M. oryzae and F. graminearum exhibited protein identities more than 50%. Pexophagy is an autophagic pathway specific for peroxisomes. A majority of ATG genes which regulate autophagy participate in pexophagy. Most of the known ATG genes present in M. oryzae, with the exception of ATG14, ATG19, ATG25, ATG30 and ATG31.To analyze their functions in peroxisome biogenesis and development of the rice blast fungus, gene knockout were processed for 12 peroxisomal biogenesis related genes. By analysis the distribution of PTS1, PTS2 and mPTS in these mutants, we found that the peroxisomal import of the PTS1- and the PTS2-containing proteins were blocked in △pex2, △pex3, △pex8, △pexl2, △pexl3, △pexl4, △pex16, △pex22 and △pex26, mPTS-containing proteins were misallocated in △pex3, △pexl6, while the disruption of PMP4, PMP47 and DJP1P do not affect PTS1-, PTS2- or mPTS-containing proteins. The transcription of possible related genes was checked in 12 mutants by quantitative PCR and showed that expression of the genes were influenced more or less in all mutants, in most cases, improved. Phenotypic analysis the mutants revealed that △pex2, △pex3, △pex8, △pex12, △pex13, △pexl4, △pex16, △pex22 and △pex26 exhibited deficiency in pathogenicity, slowed vegetative growth, reduced sporulation, decreased melanization, disordered fatty acid utilization, defective cell wall and temperature sensitivity. No remarkable influence was found in △pmp4, △pmp47 and △djplp in pathogenicity, colony morphology, growth, sporulation and fatty acid utilization. △pmp47 could also grow on middle-chain fatty acid ordinarily. The mating assay for the mutants showed that all of the 12 mutants generated perithecium normally when cross-cultured on artificial media (OMA) with wild type strain 2539 and 2539-△pmp47. However, the mutant 2539-△pmp4 mating with 2539-△pexl4 caused remarkable reduction of perithecium. This work revealed the relationship between peroxisomal biogenesis related genes and fungal development and pathogenicity, which will benefit the further understanding of pathogenesis of M. oryzae and organelles biogenesis in fungi.