| Rice blast was one of the most destructive rice disease in the world, threatened seriously the sustainable development of agricultural production. Rice blast was caused by Magnaporthe oryzae, a model fungus to study the interaction between plants and pathogens, while the pathogenic mechanism of the fungus is an important component of plant pathology. The application of genomic database pushed the study of gene function in rice blast fungus more quickly. Along with the progress of the study, the roles of various organelles, such as peroxisomes, woronin body and endoplasmic reticulum, in the development and pathogenicity of the fungus were payed more attention,Before the rice blast fungus invades host, its conidia have to degrade the stored nutrations to feed the morphogenesis of infection structures （including appressorium melanin deposition, glycerol accumulation and the formation of cell wall, etc） and supply the energy to complete the penetration progress. Lipid is an important storage in conidium. The β-oxidation and glyoxylate cycle were the main reactions in lipid degradation. Peroxisome is a kind of important organelles which enclosed the β-oxidation and glyoxylate cycle in fungus cells. Previous study showed that the number of peroxisome was varied in the progress of appressorium formation. In addition, in the process of pathogen invasion and colonization, the host produces large amount of reactive oxygen species to defend against the pathogenic fungus. The pathogen have to overcome the influence of reactive oxygen speciesfor invasion successfully.Peroxisomal matrix proteins and PMPs are synthesized in the cytoplasm. Most matrix proteins include a short peroxisomal-targeting signal （PTS）, which can be either type PTS1 or PTS2. PEX5 and PEX7 encode receptors that recognize and bind PTS1-and PTS2-containing proteins, respectively. Most PMPs have one or more membrane protein-targeting signal （mPTS） consisting of a cluster of basic residue. Pex19p is now known as both an import receptor and a chaperone for PMPs, which shuttles between the cytosol and peroxisomal membrane, binds and stabilizes newly synthesized PMPs in the cytosol, and is essential for PMP targeting and import.The study of peroxisomes biogenesis mechanism and the functions of PEX in filamentous fungi started late. Akiko et al. demonstrated the roles of peroxisome firstly in plant pathogenic fungus by analyzing the ClaPEX6 in the bean anthrax fungus （Colletotrichum lagenarium） in 2001. Compared to the studies of matrix protein receptors Pex5p and Pex7p, the functions of the peroxisome membrane protein receptor protein Pex19 were less disclosed. In plant pathogenic fungus, the machinery of PMPs import and the roles of pexl9p was still obscure. In present work, we firstly focused on thePEX19 gene in M. oryzae, MoPEX19. The findings are mainly as follows:a) Protein sequence of S. cerevisiae Scpexl9p （CAA98630.1） was used to search the Magnaporthe comparative database with the BlastP procedure. A hypothetical gene MGG00971 exhibited the most similarity to Scpexl9p. MGG00971 fully restored the growth of the Ascpexl9 mutant on oleic acid and was thus deemed as the homologue of PEX19 in M. oryzae （MoPEX19）.b) Mopexl9 was expressed at a low level and its promoter was weak. During conidial germination and appressorium development, the expression of MoPEX19 was raised gradually, with a peak at the initial germinated conidia incubated for 2 h. The localization of GFP fusion showed that Mopexl9p was distributed in cytoplasm and newly formed peroxisomes.c) Peroxisomal matrix proteins and PMPs were mislocalized in the cytoplasm of the Amopex19 mutant, as well as Hexlp, the core protein of woronin bodies. The peroxisomal structures and woronin bodies were undetected under TEM analysis.d) The Amopexl9 mutants showed reduced radial growth and aerial hyphal development. MoPEX19 was required for conidial generation and viability of M. oryzae. Deletionof MoPEX19 led to defects in lipid metabolism and decreased tolerance of reactive oxygen species （ROS）.e) MoPEX19 was required for conidial germination, appressorium development and turgor generation. Cytoplasmic leakage was found during the germination and appressorium development, and the lipid mobilization from the conidia to appressorium was blocked in of the Amopex19 mutants. The deletion of MoPEX19 also altered integrity of cell wall. The defects of Amopexl9 in appressorium affect the turgor generation in the appressorium.f) Inoculation on both rice and barley indicated that MoPEX19 was indispensable to fungal pathogenicity and the MoPEX19 deletion destroyed host infection of the rice blast fungus completely.The numbers of peroxisomes was able to increase rapidly by peroxisomal proliferation to accommodate its biochemical metabolism and adapt to cellular environment. The peroxisomal proliferation is a hot topic in recent years. PEX11 was a key factor controlling the peroxisomal proliferation. However, the mechanism of peroxisomal proliferation in plant fungal pathogens was rarely investigated. In present work, we analyzed the functions of the three members of MoPEXll family, MoPEX11A, MoPEX11B and MoPEX11C,. By phenotypic analysis of the single, double and triple knockout mutants combining with the assay of protein localization and gene expression, we demonstrated the roles of the tree genes in peroxisomal proliferation, fungus development and the functions in pathogenicity. The results are as follows:a) Three possible PEX11 genes in rice blast fungus, which exhibited different similarity levels to ScPEX11 were found by exhaustive search and sequences comparison, and assigned as MoPEX11A, B and C, respectively. MoPEX11A, B, C were separated inthreedifferent branches representing the different types of PEX11 in filamentous fungus.b) MoPEX11A, B and C were expressed at a low level in hyphae, conidium and appressorium in M. oryzae. The expression of MoPEX11A was found unregulated during germination, and up to a peak at 10 h post incubation. MoPEX11A, as well as MoPEX11C was co-localized with peroxisomal marker PTS1. MoPEX11B was distributed on the endomembrane system.c) We generated firstly the single knockout mutants of MoPEX11A, B and C by gene replacement strategies. All the mutants showed no difference on vegetative growth compared with the wild type Guy-11. The numbers and sizes of peroxisomes were altered in MoPEX11A mutants, but were unaffected in MoPEX11B and C mutants. Over-expression of MoPEX11Ainterfered the division of peroxisomes.d) MoPEX11A was required for full virulence of M. oryzae. The conidial germination, appressorial formation, appressorium turgor generation and fungal penetration were significantly reduced in AmopexllA mutant, compared with the wild-type strain Guy-11 and the recovery strain. The integrity of cell wall was also affected in MoPEX11A mutants.e) MoPEX11A, B and C were then double and triple knocked out. Test of utilization of fatty acids indicated that MoPEX11A and C played roles in lipid degradation of the fungus.f) Defects were found in the sexual reproduction of MoPEX11A mutant when cross-cultured with 2539, a strain in opposite mating type.g) The pathogenicity was decreased in all the double and triple mutants which carrying disrupted MoPEX11A locus. But the decrease levels of pathogenicity in double mutants and triple mutants exhibited on difference with MoPEX11A single knockout mutant, indicating MoPEX11A is sole one in the three genes which contribute to fungal pathogenicity.h) The expression of related genes in MoPEX11A, B and C mutants by test the transcripts abundance, including MoPEX11A, B and C themselves, other PEXs, and possible PEX11-associated genes functioning in peroxisomal proliferation.The mechanism of targeting and insertion of peroxisomal membrane proteins are more complicated compared with the import of peroxisome matrix proteins. Little was known on the roles of peroxisomal membrane proteins in fungal pathogens. In present work, we analyzed the distributions of three PMPs in M. oryzae （MoPMP4, MoPEX14, MoPMP47） by GFP fusion assay. The results as following:a) Under the control of MPG1 promoter, the GFP fusions with Mopmp4, Mopexl4 and Mopmp47 present dominantly in dot pattern in the M. oryzae cells. Of them, the fusion of GFP-Mopmp4 generated abundant fluorescence background.b) GFP-Mopexl4 trended to present in the front parts of the conidia. The fluorescence of GFP-Mopexl4 was co-localized partially with Cherry-PTS1. The fluorescent dots GFP-Mopmp47 were in larger sizes than those of GFP-Mopexl4, and GFP-Mopmp4. The fluorescence of RFP-PTS1 in co-integrated strains of RFP-PTS1 with GFP-Mopmp47 present also in larger sizes than that in RFP-PTS1 sole transformants. The reason for this phenomenon was maybe the overexpression of MoPMP47 controlled by MPG1 altered the sizes of peroxisomes.c) In wild-type strain, the mitochondrial protein Mofowl did not colocalized with PTS1 peroxisomal matrix protein. Similarly, the fluorescence of GFP-Mopexl4 did not overlay with that of mitotracker, a type of mitochondrial dye. GFP-Mopexl4 overlay partially with Hexl which located mainly at the septa region between fungal cells.d) The fusions of Mopexl4p, Mopmp47p and Mopmp4p present still mainly in dots in Δmopex5, Δmopex6 and Δmopex7 mutants, indicating that the distribution of PMPs was not disturbed by absence of MoPEX5, MoPEX6 or MoPEX7.e) Deletion of MoPEX19 blocked the transport of membrane protein Mopmp47, while Mopexl4genes was still in dots. We speculated that MoPEX14 was maybe localized at other organelles when peroxisome disappeared.In summary, we analyzed the peroxisomal membrane protein receptor gene MoPEX19 and peroxisomal proliferation related genes MoPEX11A, B and C, and found that MoPEX19 play important roles in peroxisomal biogenesis and MoPEX11A function in peroxisomal proliferation in M. oryzae. The deletion of MoPEX19 or MoPEX11A resulted in significant defects in fungal development and pathogenicity in the fungus. In addition, we analyzed the localization of three peroxisome membrane proteins, Mopex14, Mopmp47 and Mopmp4, and compared their locations with that of PTS1 protein, Hexl, mitochondria and nuclear.The findings in this work will contribute the research on organelle biogenesis in fungal cells and benefit the investigation of fungal pathogenesis. |
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