Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae§

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae § § first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:fuchenglin@zju.edu.cn †E-mail:fuchenglin@zju.edu.cn fuchenglin@zju.edu.cn State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou 310029, China State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou 310029, China Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M . oryzae . In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi. Magnaporthe oryzae Appressorium Gene expression Autophagy INTRODUCTION 1 A model fungus, Magnaporthe oryzae causing rice blast disease, employs an appressorium with a well evolved dome-shaped cellular structure, in the course of interaction with the rice host, in order to effectively penetrate different rice tissues, i.e., leaves, stems and roots (Ou, 1985 1985 ). To understand the molecular mechanisms of appressorium formation to restrain this fungal pathogen, considerable progress and efforts have been made to identify gene functions, which is necessary for the regulation of appressorium-mediated penetration events in the development and pathogenicity of M . oryzae . So far, several signal pathways related with this process have been extrapolated in M . oryzae , including the cAMP (cyclicadenylicacid) respond process, the mitogen-activated protein kinase cascade, the GTP (guanosine triphosphate)-binding (G) protein transduction pathway and the calcium-mediated signal transduction (Dean et al., 2005 2005 ; Lee and Dean, 1993 1993 ; Liu and Dean, 1997 1997 ; Nguyen et al., 2008 2008 ; Xu and Hamer, 1996 1996 ). Currently, autophagy was confirmed to be an important pathway to the turgor accumulation in appressorium (Liu et al., 2007 2007 ; Veneault-Fourrey et al., 2006 2006 ). However, molecular mechanisms in these events in this fungus have not been fully characterized. Here we report the biological roles of autophagy pathways in appressorium formation. GENES SPECIFICALLY EXPRESSED IN APPRESSORIUM FORMATION 1 We have developed a novel method for purification of total RNA from appressoria of M . oryzae . The total RNA of appressoria was isolated directly from the appressoria of this fungus inoculated on a hydrophobic surface of projection transparency film (terylene resin membrane, Gaoke, China) with Trizol reagent (Lu et al., 2005a 2005a ; 2005b 2005b ). We had successfully obtained the total RNA of M . oryzae from the appressoria incubated (from conidia) for 2, 4, 8, 12, 18, 24 and 30 h and built mature appressorium cDNA libraries (Liu et al., 2008 2008 ). Total RNA of the conidia/aerial mycelia/substrate mycelia mixture and the mature appressoria at the incubation of 23.5~24.5 h were isolated. A subtractive appressoria cDNA library, subtracted by the conidia/aerial mycelia/substrate mycelia mixture, was constructed by suppression subtractive hybridization (SSH). Among 142 ESTs (expressed sequence tags) selected, 70 were described previously and another 72 were first reported in M . oryzae . Eighty-nine percent of the ESTs (126/142) had significant matches ( P < 0.001) to known (or predicted) genes present in GenBank at the time of submission. All 142 identified genes were functionally classified: 12 (8.45%) ESTs in fat metabolism such as cytochrome P450, keto acyl reductase, acyl-CoA dehydrogenase, autophagy pathway, HMGB (high mobility group B) family and C-14 sterol reductase; 10 (7.04%) in protein modification such as serine-theronine kinase, oligosaccharyltransferase alpha subunit, and cysteine proteinase; 10 (7.04%) in nucleic acid metabolism; 8 (5.63%) in sugar metabolism; 6 (4.23%) in antibiotic metabolism; 4 (2.83%) in protein synthesis; 1 (0.70%) in cell wall; and 21 (14.79%) in other functions, whereas 70 (49.30%) ESTs were classified as having unknown function. We also used the reverse-transcript polymerase chain reaction (RT-PCR) analysis to confirm differential gene expression in appressoria and conidia/aerial mycelia/substrate mycelia mixture as indicted by SSH subtraction strategy. These results reveal differential gene expression in the appressorium stage of M . oryzae . Functional analyses of 35 novel genes related to this stage are being carried on now, and some of them have been reported, such as Mnh6 (Lu et al., 2007 2007 ), MgATG1 (Liu et al., 2007 2007 ), and Mtp1 (Lu et al., 2008 2008 ). AUTOPHAGY PATHWAY LINKED TO APPRESSORIUM MATURATION AND PENETRATION 1 Autophagy is a ubiquitous and evolutionarily conserved process that degrades and recycles long-lived proteins and organelles, occurring in all eukaryotic cells (Levine and Klionsky, 2004 2004 ). It is an intracellular, bulk degradation process in which cytosol and organelles are sequestered within double-membrane vesicles termed autophagosomes that deliver the contents to the lysosome/vacuole for degradation and recycle of the macromolecules (Yorimitsu and Klionsky, 2005 2005 ). For many years, autophagy has been presumed to be involved in cellular architectural changes that occur during differentiation and development, presumably via its role in organelle and protein turnover (Klionsky, 2005 2005 ; Levine and Klionsky, 2004 2004 ). An EST (GenBank accession No. CK828251) encoding MgATG1 , a serine/threonine kinase required for autophagy, was found in screening genes highly expressed during appressorium stage from the subtractive suppressive cDNA library of the 24-h mature appressoria. To find out the potential role of MgATG1 during appressorium formation, maturation and penetration, we isolated a full-length of MgATG1 gene from genomic DNA of M . oryzae Guy11 and appressorium stage cDNA library by long distance PCR. The 2949-bp cDNA fragment containing full coding DNA sequences (CDS) was cloned to pUCm-T (Sangon, Shanghai, China) and sequenced (GenBank accession No. DQ224381). MgATG1 in M . oryzae , containing an S_TKc domain of 256 residues, is a 982-amino acid serine/threonine protein kinase of 108 kDa. The alignment between MgATG1 of rice blast fungus and other ATG1 of other organisms shows very high homologies. Significantly, ATG1 in M . oryzae is 60% identical to CLK1 (homolog of ATG1 in yeast) in Colletotrichum lindemuthianum (Dufresne et al., 1998 1998 ), 56% to ATG1 (PDD7p) in Hansenula polymorpha (Komduur et al., 2003 2003 ), 51% to ATG1 (APG1/AUT3) in Saccharomyces cerevisiae (Matsuura et al., 1997 1997 ), and 36% to ULK1 in Mus musculus (Yan et al., 1998 1998 ), ULK1 Homo sapiens (Kuroyanagi et al., 1998 1998 ) and UNC-51 in Caenorhabditis elegans (Ogura et al., 1994 1994 ). The ∆ mgatg1 mutant of M . oryzae , in which MgATG1 gene was deleted by targeted gene replacement, shows a defect in autophagy and also exhibits defects in morphogenesis including reduction in conidiation, delay in conidia germination, and reduction in appressorium turgor. As a result, the MgATG1 disruption mutant loses its pathogenicity to two tested host plants, rice CO-39 and barley ZJ-8. These phenotypes were recovered by re-introduction of an intact copy of MgATG1 to Δ mgatg1 null mutant. Taken together, MgATG1 gene is essential in autophagy and is also required for development and pathogenicity of M . oryzae (Liu et al., 2007 2007 ). AUTOPHAGY RELATED GENES IN M . ORYZAE 1 Currently, we have isolated 21 autophagy genes homologous to yeast, which are highly conserved among other eukaryotes, including humans and plants. Disruption of other autophagy related genes, such as MgATG2 (GenBank accession No. EU984499), MgATG4 , MgATG5 (GenBank accession No. EF486491), MgATG8 , MgATG9 and MgATG18 (GenBank accession No. EU984500) involved in the autophagy pathway, influenced the capability of surviving starvation, conidiation, conidial germination, lipid turnover, and appressorium turgor generation in M . oryzae . As a result, these null mutants lose their penetration and pathogenicity into the host plants. By reintroducing the autophagy genes to their null mutants, all the defects were restored. However, targeted deletion of MgATG11 , MgATG13 and MgATG17 homologous to ATG11 , ATG13 and ATG17 in yeast respectively, did not block autophagy pathway in M . oryzae (Liu et al., 2008 2008 ), suggesting that there were different protein components involving in the autophagy machinery between two of these fungi. Taken together, clarification of the functions and network of these autophagy genes will lead to a better understanding of the role of autophagy genes in the pathogenesis of this fungus to control this fungal disease, and to the discovery of novel autophagy genes by using this model filamentous fungus. CONCLUSION 1 In this review, we summarize gene expression in appressorium formation and emphasize the autophagy pathway in M . oryzae . Studies on autophagy related genes have revealed numerous effects involved in every stage of development and differentiation in M . oryzae . 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