Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination 1 first-author 1 †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn jplu@zju.edu.cn 1 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn †E-mail:jplu@zju.edu.cnfuchenglin@zju.edu.cn jplu@zju.edu.cn College of Life Sciences, Zhejiang University, Hangzhou 310058, China College of Life Sciences, Zhejiang University, Hangzhou 310058, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae . The Mtp1 protein is 520 amino acids long and is comparable to the Ytp1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1 - GFP (green fluorescent protein) fusion expression results indicate that Mtp1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Δ mtp1 mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use. Magnaporthe oryzae Magnaporthe grisea MTP1 cDNA Gene knockout Conidial germination INTRODUCTION 1 Magnaporthe oryzae , distinguished from Magnaporthe grisea (Couch and Kohn, 2002 2002 ), is a major fungal pathogen of rice and other cereals, causing devastating rice blast disease throughout the world (Ou, 1985 1985 ). In China more than 3.8×10 6 ha of rice has been exposed to this disease every year since 1990. Much has been learned of the molecular mechanisms regulating pre- and post-penetration events in the development and pathogenicity of M . oryzae , such as those involved in appressorium formation and invasion growth (Lee and Dean, 1993 1993 ; Xu and Hamer, 1996 1996 ; Choi and Dean, 1997 1997 ; DeZwaan et al., 1999 1999 ; Clergeot et al., 2001 2001 ; Kim et al., 2005 2005 ; Zhao et al., 2005 2005 ; Veneault-Fourrey et al., 2006a 2006a ; Odenbach et al., 2007 2007 ). Among those known important genes, Pth11 is a plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues (DeZwaan et al., 1999 1999 ). Pls1 is another transmembrane protein localized in plasma membranes and vacuoles of appressoria, required for the penetration of the rice leaf by the fungal pathogen (Clergeot et al., 2001 2001 ; Veneault-Fourrey et al., 2006b 2006b ). However, the functions of most transmembrane proteins in this fungal pathogen have not been fully characterized. The plasma membrane is an interface interacting with plant cells when the pathogen penetrates into or grows in the plant. The signals outside of the cell are imported through the cell membrane to activate fungal developmental events, which may result in cell differentiation or growth (DeZwaan et al., 1999 1999 ; Gronover et al., 2001 2001 ; Dean et al., 2005 2005 ). The vacuole membrane is involved in autophagy, which is required for the pathogenicity of the rice blast fungus (Veneault-Fourrey et al., 2006a 2006a ; Liu et al., 2007 2007 ). The proteins integrated into the mitochondrial membrane are also necessary for the function of mitochondrion. The mitochondrion is an important organelle and is the site at which fatty acid β-oxidation occurs, which is necessary to synthesize glycerol in the appressorium from storage products (Dean et al., 2005 2005 ). Glycerol is necessary in order to generate turgor pressure in an appressorium, which is required for plant penetration and pathogenicity (de Jong et al., 1997 1997 ). MTP1 gene, encoding a type III integral transmembrane protein, is one of hundreds of genes isolated from the M . oryzae appressorium cDNA library in our previous study (Lu et al., 2005a 2005a ). Little is known about the functional and pathological significance of the Mtp1 protein in the rice blast fungus, or its homologues in other filamentous fungi. In this study we report the molecular structure of the MTP1 gene which encodes a transmembrane protein with 8 membrane helices, its expression profile during growth and development and its function which was elucidated through gene knockout experiments in M . oryzae . MATERIALS AND METHODS 1 Fungal strain, DNA/RNA isolation and manipulation 2 The M . oryzae wild type strain, Guy-11, and mutants (transformants and an MTP1 -knockout mutant generated from Guy-11) were cultured on complete medium (CM) plates (Talbot et al., 1993 1993 ) at 25 °C with a 14-h light and 10-h dark cycle using fluorescent lights. Studies involving a cross with strain 2539 were conducted on oat meal agar (OMA) medium (30 g oatmeal in 1 L distilled water). DNA and total RNA extraction, polymerase chain reaction (PCR), restriction digest reaction, gel electrophoresis and ligation reaction were carried out following standard procedures (Sambrook et al., 2002 2002 ). Isolation and sequence analysis of MTP1 2 The cDNA fragment containing the full coding sequence of the MTP1 gene was cloned from the cDNA library (Lu et al., 2005b 2005b ) by PCR using the primers s148RNAp1 (5′-CCACCACGGCTTCAATCCCGCGAC-3′) and s148RNAp2 (5′-GATGCACATCGGTGCAGAGCTGTC-3′), cloned into the T-vector, pUCm-T (Sangon, Shanghai, China), and sequenced on an ABI377 DNA sequencer (Invitrogen, USA). The DNA fragment containing the MTP1 gene was amplified from Guy-11 genomic DNA using the primers s148p1 (5′-GTTTGGTTAATTGTCTTCCCGGCTGTTTTG-3′) and s148p2 (5′-CACCCCCTTCCCGAGTCTTGATTTGTTGT-3′), cloned into the pCR-XL®-TOPO® vector (Clontech, USA), and sequenced. The putative protein Mtp1 was predicted for homologies by using the Blastp (Altschul et al., 1997 1997 ) and ClustalW ( http://www.ebi.ac.uk/clustalw/ http://www.ebi.ac.uk/clustalw/ ) (Thompson et al., 1994 1994 ), for subcellular localization by TargetP 1.1 ( http://www.cbs.dtu.dk/services/TargetP-1.1/ http://www.cbs.dtu.dk/services/TargetP-1.1/ ) (Emanuelsson et al., 2000 2000 ) and WoLFPSORT ( http://wolfpsort.seq.cbrc.jp http://wolfpsort.seq.cbrc.jp ) (Horton et al., 2006 2006 ), for the presence and location of signal peptide cleavage sites by SignalP 3.0 Server ( http://www.cbs.dtu.dk/services/SignalP/ http://www.cbs.dtu.dk/services/SignalP/ ) (Dyrløv Bendtsen et al., 2004 2004 ), and for protein structure by Predictprotein ( http://www.predictprotein.org/ http://www.predictprotein.org/ ) (Rost et al., 2004 2004 ). Construction of the MTP1 knockout vector 2 The knockout vector, pBS-MTP1, was constructed by inserting two flanking sequences of MTP1 gene into the pBS-HPH2 vector. pBS-HPH2, an intermediate vector, was constructed by inserting a 1344-bp Sal I- Bam HI PCR fragment containing a hygromycin phosphotransferase ( HPH ) gene expression cassette into the Sal I- Bam HI sites of the pBS(+) vector (Stratagene, USA). The HPH gene expression cassette fragment was prepared by PCR amplification from the plasmid pCB1003 (Carroll et al., 1994 1994 ) by using the primers 5′-CCggatccTGGAGGTCAACACATCAAT-3′ and 5′-CCgtcgacCTACTCTATTCCTTTGCCCTCG-3′. A 1240-bp Kpn I- Sal I downstream flanking sequence fragment of MTP1 , amplified from genomic DNA using the primers s148dnp1 (5′-AAggtaccTGCTGCTGTTACCGCGAAGGATT-3′) and s148dnp2 (5′-AAgtcgacAAGAGGCGGCGAAGAGGAAGTTAC-3′), was firstly inserted into the Kpn I- Sal I sites of pBS-HPH2 and then another 1231-bp Spe I- Not I upstream flanking sequence fragment obtained by PCR with the primers s148upp1 (5′-AAggtaccTGCTGCTGTTACCGCGAAGGATT-3′) and s148upp2 (5′-AAgtcgacAAGAGGCGGCGAAGAGGAAGTTAC-3′) was inserted into the Spe I- Not I sites of pBS-HPH2 to generate the knockout vector pBS-MTP1. The 3.5-kb Hin dIII digested pBS-MTP1 vector was used to transform protoplasts of Guy-11. Fungal transformation and identification of MTP1 knockout mutants 2 The transformation procedure, including protoplast generation, was conducted as described previously (Lu et al., 2007a 2007a ). Transformants were recovered and selected in CM with 20% (w/v) sucrose and 200 μg/ml hygromycin B (Roche Diagnostics GmbH, Mannheim, Germany). Transformants were further confirmed by passing through a second selection in the hygromycin B media. Initial identification of the knockout mutants was performed by PCR using primers internal to MTP1 (s148ckp1: 5′-CGGCGTCGTCCCTCGTGTTGG-3′ and s148ckp2: 5′-TCGCGGCGCTGGGATTCTGC-3′), resulting in amplification of an 832-bp fragment of the MTP1 gene. Putative knockout transformants identified by PCR screens were purified by single conidium isolation, and confirmed further by Southern hybridization analysis. Genomic DNAs from transformants were prepared, and cut with the Eco RV restriction enzyme for Southern blot analysis. A 547-bp DNA fragment located in the upstream flanking sequence of MTP1 gene was obtained by PCR from genomic DNA with the primers s148sb2p1 (5′-CGAAACGGCGAAGACGAA-3′) and s148sb2p2 (5′-GCCGCCGCCCGCCAACAT-3′), and used as a probe for Southern blot. Southern blot hybridizations were conducted using the DIG high prime DNA labeling and detection starter kit I (Roche Diagnostics GmbH, Mannheim, Germany). Construction and expression of the MTP1 - GFP fusion protein 2 A 1.4-kb HPH gene fragment was amplified from the plasmid pCB1003 with the primers p1 (5′-ACgcggccgcGGAGGTCAACACATCAATG-3′) and p2 (5′-CCaggcctGGTCGGCATCTACTCTATTC-3′) and cloned into the Not I- Stu I sites of pEGFP (Clontech, USA), resulting in generation of the plasmid pEGFP-HPH. A 1534-bp upstream sequence of MTP1 was amplified from Guy-11 genomic DNA with the PCR primers s148pro-p1 (5′-CTGAggatccAGCAGACGACGCGGAAGAAGAGATGAGAC-3′) and s148pro-p2 (5′-GTGTggatccCGCGGTTTAGATGCTGGTAGTATTTTG-3′) and cloned into the Bam HI site of pEGFP-HPH to generate pEGFP-MTP1. In pEGFP-MTP1, the green fluorescent protein ( GFP ) construct was under the control of the native MTP1 promoter. The promoter sequence of the NAR gene (Lu et al., 2007b 2007b ) was amplified from Guy-11 genomic DNA using the PCR primers 5′-CaggatccGGGAAGCGATTGCGTT-3′ and 5′-GGTGccatggTGTCGGTTGTGGTG-3′, and inserted into the BamH I- Nco I sites of pEGFP to produce the pEGFP-NAR vector. The GFP construct without a stop codon (TAA) under the control of the NAR promoter was amplified from pEGFP-NAR with the primers 5′-CTgtcgacGGGAAGCGATTGCGTTT-3′ and 5′-TTcccgggCTTGTACAGCTCGTCCATGC-3′, and cloned into the Sal I- Sma I sites of the pBS(+) vector to generate the vector pBS-NE. Subsequently the coding domain of the MTP1 fragment without the initiation codon (ATG) was amplified using the primers 5′-TTcccgggGCGTGGTCGAGAATTGGGAT-3′ and 5′-TTtctagaTTTCACGCAACCCGATCGCTAT-3′ from MTP1 cDNA and inserted into the Sma I- Xba I sites of pBS-NE to generate pBS-NEM. Then a 0.9-kb BAR gene fragment was obtained from pBARKS1 (Pall and Brunelli, 1993 1993 ) via PCR using the primers 5′-AatctagAGAAGATGATATTGAAGGA-3′ and 5′-AatctagaCTAAATCTCGGTGACGGGC-3′, and cloned into the Xba I site of pBS-NEM to generate pEGFP-MTP2. In the pEGFP-MTP2 plasmid, the resulting MTP1 - GFP fusion construct was under the control of the NAR promoter. The resulting vector, pEGFP-MTP1, was transformed into protoplasts of Guy-11 and selected with 200 μg/ml hygromycin B. Another resulting vector, pEGFP-MTP2, linearized with Kpn I, was transformed into protoplasts of the Δ mtp1 mutant and selected with 150 μg/ml glufosinate-ammonium (Yongnong Ltd., Zhejiang, China). Hygromycin-resistant transformants or glufosinate-ammonium-resistant transformants were individually confirmed by DNA gel blot analysis. The expression of GFP was examined using an Olympus-BX51 fluorescence microscope with a cooled CCD camera DP50 (Olympus, Japan) and Zeiss LSM-510 laser scanning microscope (Carl Zeiss, Germany). Growth characteristics of the Δ mtp1 mutant 2 Vegetative growth of both the M . oryzae Δ mtp1 mutant and Guy-11 strains was examined following a previously described procedure (Lu et al., 2007a 2007a ) in CM medium, CM-N medium (CM medium without the nitrogen source), CM-C medium (CM medium without the carbon source), CM-hyperosmotic medium (CM medium with 1 mol/L NaCl) (Talbot et al., 1993 1993 ) and YPEG medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 2% (v/v) ethanol, 3% (v/v) glycerol, 2% (w/v) agar). With the same procedure, the influences of Cu 2+ (CuSO 4 , 1.0×10 −4 mol/L) and Fe 3+ (FeCl 3 , 1.0×10 −4 mol/L) in 1/4 YG medium (0.13% (w/v) yeast extract, 0.5% (w/v) glucose, 1.5% (w/v) agar) on vegetative growth of the mutant were evaluated. Conidiation, conidial germination and appressorium development of the mutant and Guy-11 were also counted according to a previously described procedure (Lu et al., 2007a 2007a ). Appressorium turgor of the mutant was estimated using incipient cytorrhysis (cell collapse) assay (Howard et al., 1991 1991 ). Pathogenicity assays 2 The inoculation method was carried out as previously reported (Lu et al., 2007a 2007a ). Two-week-old seedlings of rice ( Oryza sativa cv. CO-39) or 8-day-old barley ( Hordeum vulgare cv. ZJ-8) were spray inoculated with conidial suspension (1×10 5 conidia/ml) and then placed in a controlled environment chamber with a 14-h light and 10-h dark cycle using fluorescent lights for 7 d (rice) or 4 d (barley). Disease severity was recorded according to the method proposed by Bonman et al.( 1986 1986 ). RESULTS 1 Isolation of MTP1 2 An EST (expressed sequence tag) (GenBank_Accn: CK828223), which encodes an M . oryzae transmembrane protein 1 (Mtp1) and is expressed in hyphae, conidium and appressorium, was identified from ESTs of a suppression subtractive hybridization cDNA library of the M . oryzae strain Guy-11 (Lu et al., 2005a 2005a ). After searching the M . oryzae database ( http://www.broad.mit.edu/ http://www.broad.mit.edu/ ), this EST was found to correspond to a hypothetical protein, MG05336.5, in strain 70-15. Then the cDNA fragment containing the full coding sequence was cloned from the Guy-11 appressorium cDNA library (Lu et al., 2005b 2005b ) using high fidelity PCR and subsequently sequenced (GenBank_Accn: DQ085779). The cDNA obtained was 1982-bp long and included 1560 bp of the coding sequence, 195 bp of the 5′ untranslated region and 227 bp of the 3′ untranslated region. The 6073-bp MTP1 genomic DNA fragment was also cloned from genomic DNA of Guy-11 by long distance PCR and sequenced (GenBank_Accn: EF525179). The nucleotide sequence of the genomic MTP1 gene contains a putative 1853-bp open reading frame with three introns that are 140-bp, 89-bp and 61-bp long. The coding sequence of MTP1 encodes a predicted mature polypeptide of 520 amino acids. This polypeptide sequence shows 46% identity in sequence to the Ytp1 protein of Saccharomyces cerevisiae (West et al., 1996 1996 ), a type III transmembrane protein, as predicted by Blastp and ClustalW (Thompson et al., 1994 1994 ; Altschul et al., 1997 1997 ) during sequence similarity searches. The Mtp1 protein also shows high similarity to many proteins in filamentous fungi, such as predicted protein (GenBank_Accn: CAP73000) in Podospora anserine (63% in identity), predicted protein (GenBank_Accn: EAA28954) in Neurospora crassa (62%) and predicted protein (GenBank_Accn: XP_001225052) in Chaetomium globosum (62%). However, these homologues in filamentous fungi have not been studied until now. Predictprotein (PHDhtm) (Rost et al., 1996 1996 ) predicted that the Mtp1 protein is likely to be a type III (polytopic) transmembrane protein with 8 membrane helices. WoLFPSORT (Horton et al., 2006 2006 ) predicted that the Mtp1 protein is a transmembrane protein, probably located at the plasma membrane. However, TargetP 1.1 (Emanuelsson et al., 2000 2000 ) predicted that Mtp1 protein is most likely to be located in the mitochondrion. SignalP 3.0 (Dyrløv Bendtsen et al., 2004 2004 ) predicted that the Mtp1 protein is not a secretory protein (by neural network). These predicted results suggested that the Mtp1 protein is a transmembrane protein, possibly located at the mitochondrial membrane or the plasma membrane. Analysis of GFP expression under the control of the native MTP1 promoter 2 A GFP expression vector (pEGFP-MTP1), under the control of the native MTP1 promoter, was constructed and transformed into the wild type M . oryzae strain. Then the differential expression pattern of the MTP1 gene at various developmental stages was analyzed using seven transformants. Observation of GFP fluorescence in transformants under an Olympus-BX51 epifluorescence microscope showed that GFP was expressed in hyphae and spores. However, it was faintly expressed in germinating spores and appressoria of M . oryzae (Fig. 1 1 , see Page 516). This implied that the MTP1 gene is more highly expressed and active in the hyphal and conidial stages than in the appressorial stage. Knockout of MTP1 2 A gene knockout strategy was employed to determine the function of MTP1 . A knockout vector, pBS-MTP1 (Fig. 2a 2a ), containing the HPH gene cassette flanked by 5′ and 3′ ends of the MTP1 gene was constructed. One MTP1 knockout mutant (F7) out of nine hygromycin resistant transformants was determined initially by PCR using primers internal to MTP1 , and then by DNA gel blot analysis, to have undergone gene deletion. In DNA gel blot analysis a 9.0-kb fragment was detected in the transformant F7 in contrast with a 4.9-kb fragment found in the wild type strain (Fig. 2b 2b ). The band shift from 4.9-kb to 9-kb showed that the MTP1 gene had been deleted in the transformant F7 (namely Δ mtp1 mutant) and that homologous recombination had occurred at a single site. Analyses of MTP1 - GFP fusion expression 2 In order to analyze Mtp1-GFP localization in a cell, a MTP1 - GFP fusion expression vector (pEGFP-MTP2), under the control of the NAR promoter, was constructed and transformed into the Δ mtp1 mutant. When observed under a normal fluorescence microscope, the GFP fluorescence was observed strongly in the hyphal cytoplasm and weakly in the vacuoles of transformants (Fig. 3a 3a ). However, under a confocal laser scanning microscope the GFP fluorescence was observed also in the hyphal vacuoles (Fig. 3b 3b ) and in conidial cytoplasm with unidentified minute dot-like structures (Fig. 3c 3c ). These observations of GFP fluorescence in transformants showed that GFP was located in the cytoplasm associated with the membrane of vacuoles or other membranes in the cytoplasm (Fig. 3 3 ). Considering the above-mentioned program-predicted results, the MTP1 - GFP fusion expression results implied that the possible locations of the Mtp1 protein are membranes on several organella in the cytoplasm. Characterization of the Δ mtp1 mutant 2 The growth in colony diameters of the Δ mtp1 mutant and the wild type strain was comparable in the CM medium, the CM-N medium, the CM-C medium and the CM-hyperosmotic medium ( P ≤0.05). The colony growth of the Δ mtp1 mutant on 1/4 YG medium was not slowed by adding Cu 2+ (CuSO 4 , 1.0×10 −4 mol/L) and Fe 3+ (FeCl 3 , 1.0×10 −4 mol/L) ( P ≤0.05), in contrast to that of the wild type strain. The colony color of the mutant and wild type strains became darker on 1/4 YG medium following the addition of CuSO 4 . Interestingly the Δ mtp1 mutant [(4.44±0.09) cm in colony diameter at 12 days post-inoculation (dpi)] grew faster than the wild type strain [(3.55±0.05) cm at 12 dpi] or MTP1 rescued strain MTG-3, a Δ mtp1 mutant in which the MTP1 gene was re-introduced via an MTP1 - GFP expression vector (pEGFP-MTP2) [(3.63±0.07) cm at 12 dpi] on the YPEG medium (complete medium containing ethanol and glycerol as a sole non-fermentable carbon source) ( P ≤0.05). Also, the colony color of the mutant was darker than that of the wild type strain in the YPEG medium. Therefore the Δ mtp1 mutant was devoid of colony growth defects in these solid media, and the MTP1 gene is not essential for the mitochondrial respiratory function. In mating experiments, the Δ mtp1 mutant formed normal perithecia with viable ascospores after 4 weeks of incubation with the opposite mating-type strain 2539. However, conidiogenesis in the Δ mtp1 mutant was reduced significantly. The mutant produced (0.70±0.05)×10 4 conidia/mm 2 on the CM medium plate, approximately close to half of that produced by the wild type strain [(1.30±0.08)×10 4 conidia/mm 2 ] or MTP1 rescued strain MTG-3 [(1.20±0.07)×10 4 conidia/mm 2 ] ( P ≤0.05). In the germination assay, conidia of the Δ mtp1 mutant were slower to germinate than those of the wild type strain (Table 1 1 ). Two hours after being placed onto plastic covers, only 21.1% of the conidia of the Δ mtp1 mutant germinated compared to 90.2% of those of wild type strain. Four hours later the rate of germ tube emergence from the Δ mtp1 mutant was still lower than that of the wild type strain. The rate of later appressorium formation in the Δ mtp1 mutant was also delayed (Table 1 1 ). Six hours post-inoculation (hpi), the rate of appressorium formation in the mutant was 66.3%, while in the wild type strain it was 86.6%. However, 24 h later the rate of appressorium formation was similar in both. In MTP1 rescued strain MTG-3, the defects of the Δ mtp1 mutant in conidium germination and appressorium formation were recovered (Table 1 1 ). To evaluate the role of MTP1 in appressorium turgor, we measured the turgor of the mature appressorium using the incipient cytorrhysis test described by Howard et al.( 1991 1991 ). The rate of collapsed appressoria in a series of glycerol solutions of varying strengths did not differ significantly between the Δ mtp1 mutant and the wild type strain ( P ≤0.05). The conidia suspension of the wild type strain Guy-11, the Δ mtp1 mutant F7 and the MTP1 -rescued strain MTG-3 (1×10 5 conidia/ml, 20 μl) was droplet inoculated on plastic coverslips at 25 °C. The experiments were repeated three times with more than 300 conidia counted each time. The same lowercase letters in each column denote that the difference estimated by the Duncan’s test ( P ≤0.05) is not significant The percentage of conidia that had elaborated a germ tube was counted at 2 hpi or 4 hpi; The percentage of conidia forming appressoria was counted at 6 hpi or 24 hpi. 2 Strain 2 Conidial germination (%) 1 1 2 Appressorium formation (%) 2 2 2 hpi 4 hpi 6 hpi 24 hpi Guy-11 90.2±1.9 a 98.5±0.2 a 86.6±5.2 a 99.3±0.4 a F7 21.1±0.6 b 91.0±1.0 b 66.3±4.0 b 96.2±1.1 a MTG-3 89.3±2.2 a 96.5±1.8 a 87.5±5.8 a 97.6±0.8 a Effect of MTP1 gene deletion on pathogenicity 2 Infection assays were performed to assess the ability of the Δ mtp1 mutant to cause disease in barley and susceptible rice. In conidial sprays no significant difference in disease symptoms could be observed on plants inoculated with either the Δ mtp1 mutant or the wild type strain (Fig. 4 4 , see Page 516). DISCUSSION 1 In this study we have cloned and sequenced the MTP1 gene, a YTP1 homologue in M . oryzae . The Mtp1 protein shares 46% amino acid identity with a previously identified S . cerevisiae Ytp1 protein (West et al., 1996 1996 ). The YTP1 gene encodes a type III transmembrane protein, which is not essential for the growth and development of yeast (West et al., 1996 1996 ). The computer programs predicted that the Mtp1 protein is a type III transmembrane protein possessing 8 membrane helices. MTP1 - GFP fusion expression experiments showed that the Mtp1 protein might be located on several membranes in the cytoplasm. The MTP1 gene is not essential for the colony growth of M . oryzae . The Δ mtp1 mutant does not confer sensitivity to starvation, Cu 2+ or Fe 3+ , and exhibits no detectable osmoregulation defect. Moreover, the Δ mtp1 mutant does not lose its mating ability to cross with the opposite mating-type strain. However, in contrast to those in Δ ytp1 mutant of yeast, which lacked a detectable growth phenotype (West et al., 1996 1996 ), the phenotypic effects displayed through deletion of the MTP1 gene in M . oryzae were marked by a reduction in conidiation and a delay in conidium germination and appressorium formation. The delay in appressorium formation may be due to a delay in conidium germination. The conidial formation and conidium germination in the rice blast fungus are a complicated process, affected by many known genes, such as a blue light receptor gene SMO1 (Hamer and Givan, 1990 1990 ), adenylate cyclase gene MAC1 (Adachi and Hamer, 1998 1998 ), two PAK kinase genes CHM1 and MST20 (Li et al., 2004 2004 ), MgWC-1 (Lee et al., 2006 2006 ), and autophagy related genes MgATG1 and MgATG8 (Veneault-Fourrey et al., 2006a 2006a ; Liu et al., 2007 2007 ). In Aspergillus oryzae or M . oryzae , conidium formation and conidial germination rely on endogenous sources for nutrient supply through autophagy (Kikuma et al., 2006 2006 ; Liu et al., 2007 2007 ). The mitochondrion is an organelle that functions at the turnover of endogenous nutrient sources and supplies adenosine triphosphate (ATP) through the tricarboxylic acid (TCA) cycle, β-oxidation and oxidative phosphorylation. Recently it was found that Mtp1 might interact with Pls1 in the appressorium of M . oryzae (personal communication, Marc-Henri Lebrun). Pls1 is a transmembrane protein (tetraspanins), only localized in plasma membranes and vacuoles of appressoria and required for the formation of the penetration peg at the base of the appressorium, probably through re-establishing cell polarity (Clergeot et al., 2001 2001 ; Veneault-Fourrey et al., 2006b 2006b ). And Mtp1 protein possibly plays its role in the function of mitochondrion or vacuole in the turnover of nutrient sources or the supply of energy sources, which could be used during the conidial formation stage and the early stage of conidial germination, or play its role through interacting with Pls1 protein at the stage of appressorium. As the Δ mtp1 mutant could use ethanol and glycerol as its sole carbon sources and produce similar appressorium turgor as the wild type strain, the Δ mtp1 mutant exhibits no defects in the mitochondrial respiratory function and fatty acid β-oxidation. Interestingly the Δ mtp1 mutant seems to use ethanol and glycerol more readily than the wild type strain, as it grows faster than the wild type strain in non-fermentable carbon sources. 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