- Email: [email protected]
MoMon1 is required for vacuolar assembly, conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae
Research in Microbiology 164 (2013) 300e309 www.elsevier.com/locate/resmic
MoMon1 is required for vacuolar assembly, conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae Hui-Min Gao a, Xiao-Guang Liu a, Huan-Bin Shi a, Jian-Ping Lu b, Jun Yang c, Fu-Cheng Lin a,c, Xiao-Hong Liu a,* a
State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou 310058, China b College of Life Sciences, Zhejiang University, Hangzhou 310058, China c China Tobacco Gene Research Center, Zhengzhou Tobacco Institute of CNTC, Zhengzhou 450001, China Received 29 February 2012; accepted 26 December 2012 Available online 29 January 2013
Abstract Mon1 protein is involved in cytoplasm-to-vacuole trafficking, vacuolar morphology and autophagy, and is required for homotypic vacuole fusion in Saccharomyces cerevisiae. Here we identify MoMON1 from Magnaporthe oryzae as an ortholog of S. cerevisiae MON1, essential for the morphology of the vacuole and vesicle fusion. Target gene deletion of MoMON1 resulted in accumulation of small punctuate vacuoles in the hypha and hypersensitivity to monensin, an antibiotic that blocks intracellular protein transport. The DMomon1 mutant exhibited significantly reduced aerial hyphal development and poor conidiation. Conidia of DMomon1 were able to differentiate appressoria. However, DMomon1 was non-pathogenic on rice leaves, even after wound inoculation. In addition, DMomon1 was slightly hypersensitive to Congo red and SDS, but not to cell wall degrading enzymes, suggesting significant alterations in its cell wall. The autophagy process was blocked in the DMomon1 mutant. Taken together, our results suggest that MoMON1 has an essential function in vacuolar assembly, autophagy, fungal development and pathogenicity in M. oryzae. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Conidiation; Vacuole; Autophagy; Virulence; Magnaporthe oryzae
1. Introduction The vacuole/lysosome is a terminal destination for ERmediated protein traffic in eukaryotic cells. Vesicle docking and fusion with a vacuole rely on a SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)-dependent reaction. As the first step, chaperones (NSF/Sec18p and -SNAP/Sec17p et al.) disassemble the CisSNARE complex (when on the same membrane) from the vacuole membrane; then, the C-Vps/HOPS (homotypic fusion and vacuolar protein sorting) complex is released from the SNAREs and reacts with rab GTPase Ypt7 (Ypt7p is required for all membrane fusion steps in which the vacuole is
* Corresponding author. Tel.: þ86 571 88982291; fax: þ86 571 88982183. E-mail address: [email protected] (X.-H. Liu).
involved) to accomplish tethering; finally, docking and fusion occur. The interaction of Mon1 and Ccz1 as a stable protein complex is required for the docking of SNARE complexes (Wang et al., 2003). The Mon1eCcz1 complex is not only essential for SNARE pair association during homotypic vacuole fusion, but also in other trafficking pathways to the vacuole (Wang et al., 2002). In yeast, the Mon1eCcz1 complex is regarded as a guanine nucleotide exchange factor (GEF) of Ypt7, which triggers endosomal maturation by activating Ypt7 in late endosomes (Nordmann et al., 2010). In yeast, MON1 was first identified as a gene involved in sensitivity to brefeldin A and monensin (Muren et al., 2001). Mon1 was classified into the SAND family despite its weak similarity with proteins from this family. MON1 orthologous genes were identified in major eukaryotes such as Fugu rubripes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana (Cottage et al., 2001; Hoffman-Sommer
0923-2508/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2013.01.001
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
et al., 2009; Nordmann et al., 2010; Wang et al., 2002, 2003). Mon1 interacts with Ccz1 during fusion of vesicles and the vacuole membrane. Deletion of MON1 in yeast results in mutants with severe vacuolar protein-sorting defects. In C. elegans, MON1 orthologous gene SAND-1 is required for Rab7 function at the early and late endosomes and it regulates the cellular levels of both Rab5 and Rab7 (Poteryaev et al., 2007). Magnaporthe oryzae is a filamentous ascomycete fungus which is an excellent model organism for studying plantepathogen interactions (Talbot, 2003; Valent, 1990). The fungus infects its host through a specialized infection structure called appressorium that attaches tightly to the plant surface and directs penetration of the fungus into the host tissue by breaching the cuticle and plant cell wall (Ebbole, 2007; Talbot, 2003; Wilson and Talbot, 2009). Despite its importance, the M. oryzae ER to vacuole traffic is poorly understood. Recently, a P-type ATPase (APT2) was determined to be essential for exocytosis during plant infection (Gilbert et al., 2006). Two putative Hsp70 family proteins, Kar2 and Lhs1, were found to function as chaperones for protein translocation and maturation in the ER (Yi et al., 2009). The LHS1 gene is necessary for conidiogenesis and pathogenesis. MoSec22 and MoVam7, the orthologs of yeast SNARE proteins Sec22 and Vam7, play an important role in vacuolar morphogenesis, mycelial growth, sporulation, appressorium formation and pathogenicity (Dou et al., 2011; Song et al., 2010). MGG_12832 protein shares overlapping functions with Snx41 and Atg20. MoSnx41 associates with autophagosomes and is required for pathogenicity. The Snx41-GFP is present as puncta or short tubules that partially associate with autophagosomes or are distributed on the surface of the vacuoles during vegetative and pathogenic development in M. oryzae (Deng et al., 2012). Despite accumulated knowledge of the yeast Mon1, little is known about Mon1 proteins from filamentous fungi. In this study, we investigated the possible functions of MoMON1 in M. oryzae and showed that its deletion led to important defects in aerial hyphal development, sporulation, autophagy, appressoria formation and plant pathogenicity. 2. Materials and methods 2.1. Fungal strains and culture conditions The wild-type strain Guy11 and all mutants described in this study were maintained and cultured on complete medium plates (CM) at 25 C with standard growth and storage procedures (Talbot et al., 1993). MM media (6 g NaNO3, 0.52 g KCl, 0.52 g MgSO4, 1.52 g KH2PO4, 10 g glucose, 0.5% biotin) was used to characterize growth of the strains. To observe sexual reproduction, strains were cultured on oatmeal agar medium (OMA) (30 g oatmeal in 1 l distilled water) for 4 weeks. 2.2. DNA/RNA manipulation and quantitative RT-PCR Most general molecular biology techniques for nucleic acid analysis, including cloning, restriction digest, gel electrophoresis, ligation reaction and DNA gel blot hybridization
301
analysis were performed according to standard protocols (Sambrook et al., 2002). For genomic DNA isolation and RNA extraction, cultures were grown in liquid CM at 25 C for 3e4 days with 150-rpm shaking. For quantitative RT-PCR, mycelia were collected and subjected to ER stress treatments, such as DTT (10 mM) for 30 min and tunicamycin (10 mg/mL) for 1 h. The mycelia were collected and quickly frozen in liquid N2 for RNA extraction. Total RNA of the above mixture was isolated by a Trizol method following the manufacturer’s procedure (Molecular Research Center, Inc., USA). cDNA reverse transcribed from 500 ng of total RNA with random 6 mers using the SYBRÒ ExScriptTM RT-PCR kit (TaKaRa Bio Co Ltd, Dalian, China) was diluted to 1:3 with nuclease-free water. Real-time PCR reactions were performed in 20 ml volume containing 200 nM of each primer, 0.5 ml of cDNA and 10 ml of 2 SYBRÒ Premix Ex Taq (TaKaRa Bio Co Ltd, Dalian, China). Real-time PCR was run on a MastercyclerÒ ep realplex (Eppendorf, USA). The cycling conditions were: 30 s at 95 C followed by 40 cycles of 5 s at 95 C and 31 s at 60 C. Fluorescence signal data were collected during the 60 C phase of each cycle. Melt curves from 72 C to 60 C in 0.5 C increments, measuring fluorescence at each temperature, were collected for all samples following the last cycle. To analyze relative abundance of transcripts with the 2DDCt method (Livak and Schmittgen, 2001), the average threshold cycle (Ct) was normalized by b-tubulin for each condition as 2DCt. Fold-changes were calculated as 2DDCt. The primer pairs used for quantitative RT-PCR are listed in Supplemental Table 1. The PCR reactions were repeated at least twice independently with three replicates, and a representative set of data was presented. 2.3. Isolation and sequence analysis of MoMON1 A full-length cDNA fragment of MoMON1 was isolated from the cDNA library of Guy11 (Lu et al., 2005) using primers Mp1 and Mp2; PCR primer pairs were designed based on the M. oryzae genome database (http://www.broadinstitute. org/annotation/genome/magnaporthe_comparative/ MultiHome.html). cDNA of MoMON1 was then cloned into the pUCm-T T-vector (Sangon, Shanghai, China) and verified by sequencing. The ClustalW program was used for amino acid sequence alignments and the calculated phylogenetic tree was established using MEGA 4.0 Beta program. 2.4. Disruption of MoMON1 and complementation of the DMomon1 mutant To replace the gene, two flanking sequences of MoMON1 were amplified from the Guy11 genomic DNA and inserted into the pBS-HPH1 vector (Liu et al., 2007). A 1.02-kb upstream of MoMON1 was amplified with primers Mupp1 and Mupp2 and cloned between the XhoI and SalI sites on the pBS-HPH1 vector to generate pBS-HPH1-up. Then, a 1.01 kb downstream of MoMON1 was amplified with primers Mdwp1 and Mdwp2 and inserted into the PstI and XbaI sites of pBS-HPH1-up to
302
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
generate the pBS-MoMon1 vector. Finally, the specific fragment containing the MoMON1 upstream fragment, hygromycin gene and downstream fragment were dissected with XhoI/XbaI from the pBS-MoMon1 vector inserted into the corresponding sites of pCAMBIA1300 to generate the final deletion vector p1300-MON1. The replacement vector was introduced into Agrobacterium tumefaciens strain AGL-1 and then transformed into M. oryzae Guy11 by the ATMT method to generate MoMON1 null mutants, as described previously (Rho et al., 2001). Candidate transformants were selected by 200 ug/ml hygromycin B and were checked by PCR using primers Myzp1 and Myzp2. Then, genomic DNA of candidate transformants was extracted under standard conditions. Southern blot were carried out according to the protocol of the digoxigenin (DIG) high prime DNA labeling and detection starter kit I (Roche, Germany). For Southern hybridization analysis, the genomic DNA was digested with ApaI and separated in a 0.7% agarose gel. The labeled probe was amplified from genomic DNA of Guy11 using primers Mdwp1 and Mdwp2. For complementation assays, the 4.38-kb PCR product contained a 1.5-kb upstream sequence, a full-length MoMON1 gene coding region and a 0.98-kb downstream sequence, was amplified from the Guy11 genomic DNA using primers Mcop1 and Mcop2 and cloned into the pGEM-T easy vector (Promega, UK). The EcoRI/XbaII-digested PCR fragment was then ligated to the EcoRI/XbaI site in pCAMBIA1300X-SUR to obtain p1300SUR-MON1; the selectable marker SUR gene (sulfonylurea-resistant gene) was obtained from the pCB1532 vector and cloned into the EcoRV/EcoRI site of pCAMBIA1300X. The AGL-1 strain with the complementation vector was transformed using ATMT (A. tumefaciens-mediated transformation), by co-culture with the DMoMON1 strain. Transformants were selected on DCM (yeast nitrogen base without amino acid 1.7 g/l, NH4NO3 2 g/l, L-asparagine 1 g/l, glucose 10 g/l, Na2HPO4 used to adjust pH to 6.0) supplemented with 100 mg/mL sulfonylurea. 2.5. Phenotypic analysis For conidia production, strains were grown on CM medium at 25 C with a 12-h photophase for 7 days. Conidia were harvested from three mycelial discs (1 cm in diameter) and suspended in 3 ml sterile distilled water by vortexing and counted with a hemocytometer under a microscope. For growth, mycelial plugs (5 mm in diameter) were transferred from 7-day-old CM plates and grown on other fresh medium (OMA, CM with agents), then incubated at 25 C with a 12-h photophase. The radial growth of vegetative mycelia was measured after 4 days and 8 days. Conidial germination and appressorium formation were measured on plastic cover slips at a concentration of 2 104 conidia/ml in sterile distilled water and incubated at 25 C for 2e48 h. Conidial germination and appressoria formation were observed and counted under the microscope. ID50 values (effective concentration to inhibit fungal growth by 50%) were determined for each compound by calculating the percent inhibition [(mean colony diameter on
unamended media mean colony diameter on amended media)Omean colony diameter on media] and subjecting data to probit analysis software. All data were valid with P < 0.05 and efficient >95%.
compoundunamended using DPS relative co-
2.6. Pathogenicity assay Pathogenicity assays were performed as described previously (Liu et al., 2007). For spray infection assays, the suspension with 2 104 conidia/ml was sprayed wellproportioned onto plant leaves using an artist’s airbrush (Badger Co., IL); 2-week-old seedlings of rice (Oryza sativa cv. CO-39) were used. Inoculated rice were placed in a dew and dark chamber at 22 C for 48 h and then transferred to the growth chamber with a photoperiod of 12 h. Lesion formation was examined after 7-day incubation. For the cut leaf assay, mycelial plugs (10-day-old, 1 cm in diameter) were removed from CM medium and deposited onto the upper surface of the isolated barley cut leaves, and then incubated at 25 C with a 12-h photophase for 4 days; 8-dayold barley (Hordeum vulgare cv. ZJ-8) seedlings were used. The root infection assay was carried out as described previously (Dufresne and Osbourn, 2001). 2.7. Electron microscopy Conidia collected from 10-day-old mycelia were cultured at 28 C for 48 h in CM liquid medium with continuously shaking at 150 rpm. The mycelia were collected, washed in sterile distilled water to the full, then transferred to distilled water with 2 mM phenylmethylsulfonyl fluoride (PMSF) and incubated at 28 C for 4 h with shaking at 150 rpm. The mycelia cultured in CM liquid medium for 48 h and PMSFinduced mycelia were collected respectively, and the samples were fixed and postfixed as described previously (Liu et al., 2007). After that, the samples were examined under a JEM-1230 electron microscope (JEOL, Tokyo, Japan) operating at 70 kV. 3. Results 3.1. Identification and deletion of the MoMON1 gene in M. oryzae The protein sequence of yeast Mon1 was used to search the M. oryzae genome database using BlastP, identifying MGG_05755.7 as the most similar protein (30% identity). MoMON1 was designated. Then the full coding sequence (CDS) was cloned from Guy11 using high-fidelity PCR and subsequently sequenced (GenBank no.: JN845639). The CDS obtained is 1893 bp in length and putatively encodes a protein of 630 amino acids. The MoMon1 protein sequence from Guy11 was identical to MoMon1 from 70 to 15 (MGG_05755.7) and displayed a high level of similarity to Mon1 from other fungi, such as Aspergillus oryzae (XP_001819506.2, 91% identity), Gibberella zeae
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
303
(XP_387259.1, 96% identity) and Trichoderma reesei (EGR50254.1, 93% identify). A phylogenetic tree of Mon1related proteins identified in different fungal genomes is shown in Fig. 1. To determine the molecular function of MoMon1 in M. oryzae, we constructed a deletion mutant by targeted gene replacement using ATMT (Fig. S2A). Southern blot analyses were performed to confirm single-copy genomic integration and exclude additional ectopic integrations. An approximately 7.5 kb band was detected in mutants m-12 and m-35, in contrast to an approximately 3.9 kb band in wild type Guy11 (Fig. S2B). Two mutants (M-12 and M-35) showed comparable phenotypes and one (M12) was chosen for further studies. Complementation assay was carried out and transformant Com-12, which contained a full-length gene copy of MoMON1 (data not shown), was selected for further studies.
plates. Some conidia exhibited abnormal morphologies compared to wild type or Com-12 (Fig. 2C). Conidiophores and conidial formation were further analyzed. Small amounts of hyphae, conidiophores and conidia were observed in the DMomon1 mutant at 24 h post-conidial induction after 8-day incubation (Fig. 2D). Conidial germination and appressorium formation rates of the DMomon1 mutant were assessed. The conidial germination rate of the mutant was similar to the wild type, but only 50% of these conidia differentiated appressoria, compared to 99% in the wild type. Our results showed that MoMON1 is required for full mycelial growth, sporulation and appressorium formation.
3.2. MoMON1 affects vegetative growth, conidiogenesis and appressorium formation
Because the mycelial growth of the MoMON1 mutant was interrupted, we further monitored the effects of various cellwall-perturbing agents on the DMomon1 mutant. Mycelial growth was measured on CM plates amended with compounds (SDS, Congo red and caffeine) that cause cell wall stress, respectively (Choi et al., 2009). Mycelial growth of the DMomon1 mutant and wild-type strain was reduced on CM containing Congo red or SDS, and the DMomon1 mutant displayed sensitivity to Congo red and SDS. Mycelial growth of the DMomon1 mutant was reduced on CM containing Congo red or SDS compared to the wild type (Fig. 3). The wild type ID50 value for SDS was similar to the value for the DMomon1 mutant, but the wild type ID90 value for SDS was 2.3-fold higher than that for the DMomon1mutant (Table 1). The ID50 value of Congo red for the wild type was about 2.5fold higher than that of the DMomon1 mutant (Table 1). Calcofluor white (CFW) was used to probe cell wall integrity. It was mostly distributed at the septa and tips where chitin, one of the main components of the fungal cell wall, was actively synthesized. There was no significant difference between Guy11, the mutant and Com-12 (Fig. S4). The enzymatic activity of glucanex was assayed using culture filtrates from the wild type and the mutant grown in CM liquid media. No significant difference in the rate of protoplast release was found at 30 min (Fig. S5). It is reported that caffeine has an effect on a variety of processes, including changes in cell morphology in yeast (Kucharczyk et al., 1999); we further tested the effect of caffeine in M. oryzae. As illustrated in Fig. 3, the DMomon1 mutant was only slightly more sensitive to caffeine than the wild type (wild type ID50 1.3-fold higher than DMomon1 value, Table 1). Since Mon1 in yeast was initially identified during screening of deletion strains sensitive to monensin, which interferes with intracellular trafficking, we tested the sensitivity of the DMomon1 mutant to monensin in M. oryzae. The DMomon1 mutant colonies were unable to extend on CM media with 5 ug/ml monensin, while the wild-type strain colonies continued to slowly grow (Fig. 3), suggesting that deletion of MoMON1 leads to hypersensitivity to monensin. The ID50 value of monensin for Guy11 was about 2-fold higher than that of the DMomon1 mutant (Table 1). The findings above indicate that deletion of
The morphology of the strains was monitored in different media. The DMomon1 mutant showed reduced vegetative growth in comparison to wild-type strain Guy11 and rescued strain Com-12 on CM, OMA and MM medium (Fig. 2A). The DMomon1 mutant produced sparse aerial hyphae, in contrast with the dense aerial hyphae of the wild-type strain and rescued strain Com-12; especially in OMA and MM medium plates, the DMomon1 mutant lacked aerial hyphae (Fig. 2A). Deletion of MoMON1 had a serious effect on mycelial growth, with slight decrease in mature hyphae (Fig. S3). We found that the DMomon1 mutant scarcely produced spores (w0.5%) relative to the wild-type strain on CM plates (Fig. 2B) and no spore was produced on OMA or MM medium
Fig. 1. Phylogenetic tree constructed using MEGA4.0 beta program neighborjoining tree with 1000 bootstrap replicates of phylogenetic relationships between Mon1 homologs in eukaryotes. The compared sequences were from M. oryzae (MoMon1), S. cerevisiae (CAA96832.1), N. crassa (XP_959164.2), M. musculus (NP_766603.1), H. sapiens (NP_115731.2), D. melanogaster (NP_608868.1), Gibberella zeae (XP_387259.1), Trichoderma reesei (EGR50254.1), Neurospora tetrasperma (EGO52340.1), Fusarium oxysporum (EGU75269.1), Aspergillus clavatus (XP_001272699.1), Phaeosphaeria nodorum (XP_001802326.1) and Chaetomium globosum (XP_001220171.1). The numbers at branch nodes are bootstrap values.
3.3. MoMON1 is hypersensitive to vesicular transport inhibitors and displays cell wall defects
304
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
Fig. 2. Growth and sporulation of the DMomon1 mutant. (A) Guy11, the DMomon1 mutant, and DMomon1 complement strain Com-12 were grown on CM, OMA and MM medium for 8 days. The DMomon1 mutant displayed extremely sparse aerial hyphae, especially on MM medium plates. (B) Few conidia were collected in the DMomon1 mutant, in contrast to Guy11 and Com-12. Error bars represent standard deviations. Reduction is significant (P ¼ 0.01) according to Duncan’s multiple range test. (C) Morphology of the conidia. Conidia were collected from colonies of strains on CM media. The DMomon1 mutant showed abnormal conidia. Scale bar ¼ 10 mm. (D) Development of conidia on conidiophores was observed under a light microscope 24 h after induction of conidiation under cover slips. Few conidia and conidiophores developed in the DMomon1 mutant. Scale bar ¼ 50 mm.
the MoMON1 in M. oryzae altered cell wall integrity and led to higher sensitivity to cell wall disturbing agents and vesicular transport inhibitors. 3.4. MoMON1 is involved in the protein vesicular traffic To characterize whether expression of vacuole trafficking and ER-related genes such as PDE1, MgAPT2, LHS1, KAR2, MoVAM7, MoSEC22 and MoSNX41 was affected by the MoMON1 mutation, quantitative RT-PCR was performed
using RNA extracted from cultures of the wild type and the mutant after treatment with inhibitors. Cultures of the wild type and mutant were treated with 10 mM DTT (ER stress inducer) or 10 mg/mL tunicamycin (protein glycosylation and traffic inhibitor). Expression data on marker genes were assessed under these conditions in the DMomon1 mutant compared to the wild type (Fig. 4). Upon DTT treatment, the expression levels of MoVAM7, MoSEC22, LHS1 and KAR2 were strongly decreased compared to their upregulation in the wild type (Fig. 4A). This was also observed for MoSEC22,
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
305
vacuole. In the DMomon1 mutant, neutral red staining was localized in small vacuoles, while it stained large globular vacuoles in the wild type (Fig. 5A). FM4-64 staining confirmed that the DMomon1 mutant accumulated numerous small vesicles (Fig. 5B). Ultrastructural analysis using transmission electron microscopy showed that the hyphae of DMomon1 accumulated a large number of vesicles, while typical large vacuoles were observed in Guy11 and Com-12 (Fig. 5C). Based on previous reports, fusion of autophagosomes with the vacuole and subsequent breakdown of the singlemembrane autophagic body in the vacuole is necessary for the complete autophagy pathway (Levine and Klionsky, 2004). However, our results showed that, when cultured in sterile distilled water (starvation-induce) in the presence of 2 mM PMSF for 4 h, no autophagic bodies were observed in the lumen of vacuoles in DMomon1 mutant hyphal cells (Fig. 5C). Thus, we concluded that the autophagy pathway was blocked
Fig. 3. Effects of cell-wall-disorganizing agents upon the DMomon1 mutant. The DMomon1 mutant, Guy11 and Com-12 were incubated on CM plates supplemented with various stress inducers at 28 C for 8 days. Growth of the strains in media supplemented with SDS (0.01%), Congo Red (200 mg/ml), caffeine (2 mM), and monensin (5 ug/ml).
LHS1 and KAR2 upon tunicamycin treatment (Fig. 4B). These results suggest that the absence of MoMON1 impairs the transcriptional response induced by ER stress.
3.5. MoMON1 is required for the autophagy pathway Cytological analyses showed that DMomon1 accumulated numerous vesicles that might be small vacuoles. Vital dyes neutral red and FM4-64 were used to stain vesicles of the wild type and the mutant. In normal cells of Guy11 or Com-12, neutral red was quickly internalized, enabling staining of the
Table 1 ID50 and ID90 values for cell-wall-perturbing agents. Strains ID50 ID90
Guy11 DMomon1 Guy11 DMomon1
Congo red (mg/ml)
SDS (%)
Caffeine (mM)
Monensin (ug/ml)
0.327 0.138 4.930 3.411
0.010 0.008 0.037 0.016
3.742 2.927 8.057 5.433
0.310 0.165 30.932 8.185
Fig. 4. Effect of DTT and tunicamycin on expression of ER-related genes in the wild type and DMomon1 mutant. Mycelia were grown in lipid complete medium for 2 days and then subjected to treatment with 10 mM DTT (A) or 10 mg/mL tunicamycin (B). Expression data were normalized using the btubulin gene and calibrated against the profile of mycelia grown without treatment. Error bars represent standard deviations and asterisks represent significant differences among stains tested. Reductions were significant (P ¼ 0.01 or P ¼ 0.05) according to Duncan’s multiple range test.
306
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
in the DMomon1 mutant under starvation conditions in M. oryzae. 3.6. MoMON1 plays a key role in pathogenicity Pathogenicity assays of the DMomon1 mutant were performed on two susceptible hosts (rice and barley). Since the DMomon1 mutant produced few conidia, we used weak inocula (2 104 conidia/ml) for spray inoculation. Results showed that the wild-type strain and the MoMON1 complementary strain caused visible brown and punctuated lesions on rice leaves, whereas the DMomon1 mutant caused no macroscopic lesions (Fig. 6A). Mycelial plugs of DMomon1 inoculated onto detached barley did not cause disease symptoms, while the wild-type strain induced susceptible lesions. Although the cuticle of detached barley was damaged, there were no visible lesions when mycelial plugs of DMomon1 were inoculated (Fig. 6B). In the root infection assay, no visible brown lesions developed on rice roots when the DMomon1 mutant mycelial plugs were inoculated (Fig. 6C). In contrast, the wild-type strain and the MoMON1 complement strain caused typical rice blast lesions in the same tissues after 3 weeks of inoculation. Taken together, these data suggested that MoMON1 is indispensable for the pathogenicity of M. oryzae. 4. Discussion In yeast, Mon1 is a monensin hypersensitivity protein required for nearly all ER traffic pathways, as well as in ER for the vacuole protein sorting pathway (Wang et al., 2002, 2003). We isolated MoMON1, an M. oryzae ortholog of yeast MON1. Targeted deletion of MoMON1 has a strong effect on sporulation, vacuolar morphology, autophagy and pathogenicity in rice and barley. Conidiation is one of the key stages of the rice blast fungus for carrying out the infectious cycle. The DMomon1 mutant is defective in aerial hypha formation and conidiogenesis, and most of the conidia are abnormal. MPS1 is a mitogen-activated protein-kinase-encoding gene responsible for regulation of cell wall growth under membrane stress conditions (Xu et al., 1998). Similarly to the DMomon1 mutant, the Dmps1 mutant also showed dramatically reduced aerial hypha formation and conidiation (Xu et al., 1998). However, progressive autolysis was observed in the colony of the Dmps1 mutant in contrast to the sparse aerial hypha of the DMomon1 mutant.
Fig. 5. Fragmented vacuole in the hyphae of the DMomon1 mutant. (A) Neutral red staining of vacuoles. Numerous small vacuoles are observed in theDMomon1 mutant, while large globular vacuoles are observed in the wild type and Com12. Scale bar ¼ 10 mm (B) FM4-64 staining of membranes of neutral vacuoles in Guy11 and Com-12. Small vacuoles were found in the
DMomon1 mutant. Strains were grown for 24 h on CM-overlaid microscope slides before staining. Scale bar ¼ 10 mm. (C) Blocking of vacuole-related fusion pathways in the DMomon1 mutant. Left side: mycelia cultured at 28 C for 48 h in CM liquid medium continuously shaken at 150 rpm. Right side: starvation-induced mycelia, where autophagy pathways were triggered. In the DMomon1 mutant, numerous smaller vacuoles were presented under nutrient and starvation conditions; the organelle and autophagosomes were detected in vacuoles of mycelia PMSF-induced from Guy11 and Com-12 (indicated by the arrow), whereas no autophagic body was evident in DMomon1 mutant vacuoles.
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
307
Fig. 6. Loss of pathogenicity of the DMomon1 mutant. (A) Rice seedlings aged two weeks were inoculated by spraying 2 104 conidia/ml of Guy11, DMomon1 mutant and Com-12 individually with 0.2% gelatin as control. After 7-day inoculation, lesion formation on rice leaves was observed. (B) Disease symptoms on cut leaves of barley (intact and wounded) inoculated with mycelial plugs from Guy11 and the DMomon1 mutant and Com-12, individually with agar as control. Typical leaves were photographed 4 days after inoculation. (C) Root infection assay. Arrows show necrotic lesions, the DMomon1 mutant, Guy11, undisposed rice as control and Com-12.
MoSEC22 and MoSNX41 are required for sporulation in M. oryzae (Deng et al., 2012; Song et al., 2010). The DMomon1 mutant has a defect in conidiation, but the few conidia were able to develop appressoria at the tip of the germ tube. Unlike the DMovam7 mutant, conidia could not differentiate appressoria (Dou et al., 2011). Taken together, we reasoned that MoMON1 plays a role in formation of conidia and appressoria. Fungal cell walls have a complex structure, and many stresses or developmental processes, as well as plant infection, can induce their remodeling (Cabib et al., 2001). Our results showed that mycelial growth of the DMomon1 mutant decreased in the presence of caffeine, SDS and Congo red. These observations are in accordance with earlier reports on
MoVam7 and Mosec22 (Dou et al., 2011; Song et al., 2010). The DMomon1 mutant was found to be insensitive to cellwall-digesting enzymes. Unlike Dmps1, it produced protoplasts at a faster rate than Guy11 under treatment of cellwall-digesting enzymes (Xu et al., 1998). Interestingly, Dmosec22 was resistant to lysing enzymes (Song et al., 2010). These results indicate that deletion of MoMON1 influenced the composition and/or structure of the cell wall, and the resulting mutant showed differences from those with deletion of other genes, such as MPS1 and MoSEC22. qRT-PCR analyses showed that genes normally upregulated in response to ER stress (MoSEC22, LHS1, KAR2, MoVAM7 Yi et al., 2009) were downregulated in theDMomon1 mutant
308
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
compared to the wild type. These results suggest that MoMON1 is required for the transcriptional response induced by ER stress. The hypersensitivity of the DMomon1 mutant to monensin, an inhibitor of vesicle trafficking in eukaryotes (Tartakoff, 1983), strongly suggests that this mutant is altered in intracellular protein trafficking. In yeast, the Mon1 protein forms a stable complex with the Ccz1 protein which binds to the vacuole membrane. The Ccz1eMon1 complex is known to function at the tethering/ docking stage of vacuole membrane fusion or homotypic vacuole fusion, and deletion of either Ccz1 or Mon1 also resulted in a fragmented vacuole phenotype in Saccharomyces cerevisiae (Wang et al., 2002, 2003; Weber et al., 2001). Likewise, the Mon1 ortholog SAND-1 is required for late steps of endocytosis in C. elegans. The sand-1 (or552) mutants displayed abnormal receptor-mediated endocytosis (Poteryaev and Spang, 2005; Poteryaev et al., 2007). Additionally, in Aspergillus nidulans, disruption of the VpsA gene essential for vacuolar biogenesis leads to fragmented vacuoles (Tarutani et al., 2001); in M. oryzae, null mutants of the SNAREencoding genes MoVAM7 and MoSEC22 also displayed small fragmented vacuoles (Dou et al., 2011; Song et al., 2010). Furthermore, cytological analyses revealed that numerous small vacuoles accumulated in the DMomon1 mutant. These results suggest that MoMON1 is required for fusion of vesicles and vacuoles, as observed in yeast. It has been reported that the autophagy pathway is a common and evolutionarily conservative process that delivers proteins and organelles to the vacuole (Mizushima, 2007). The autophagy pathway that delivers organelles and cytoplasmic content to the vacuole has been shown to be essential for fungal development and pathogenicity (Bartoszewska and Kiel, 2012; Liu et al., 2012). We showed that hypha of the DMomon1 mutant displayed fragmented vacuoles and that the autophagic pathway was blocked under either nutrient or starvation conditions. Similarly, the autophagic gene MoATG1 resulted in a reduction in appressorium turgor and loss of pathogenicity (Liu et al., 2007). The MgATG8 null mutant is also impaired in autophagy and pathogenicity (VeneaultFourrey et al., 2006). In A. oryzae, the DAoatg8 mutant also had defects in the formation of conidia and aerial hyphae (Kikuma et al., 2006). However, the morphology of the vacuole was not altered in DMgatg1, DMgatg8 and DAoatg8 mutants. Taken together, our studies suggest that autophagy was blocked in the DMomon1 mutant and resulted from defects in vacuole-related fusion pathways. In summary, we have identified and characterized MoMON1, a gene encoding a vacuolar fusion protein in M. oryzae. MoMON1 plays an important role in the differentiation processes of M. oryzae, including intracellular transport and development in vegetative growth, conidiogenesis, appressorium formation and pathogenicity toward plants. Acknowledgments We thank the anonymous reviewers for their careful and thoughtful reviews and helpful advice.
This work was supported by grants (No. 30925029 and 31000077) funded by the National Natural Science Foundation of China. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.resmic.2013.01.001. References Bartoszewska, M., Kiel, J.A., 2012. The role of macroautophagy in development of filamentous fungi. Antioxid. Redox Signal 14, 2271e2287. Cabib, E., Roh, D.H., Schmidt, M., Crotti, L.B., Varma, A., 2001. The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J. Biol. Chem. 276, 19679e19682. Choi, J., Kim, Y., Kim, S., Park, J., Lee, Y.H., 2009. MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae. Fungal Genet. Biol. 46, 243e254. Cottage, A., Edwards, Y.J.K., Elgar, G., 2001. SAND, a new protein family: from nucleic acid to protein structure and function prediction. Comp. Funct. Genom. 2, 226e235. Deng, Y.Z., Qu, Z., He, Y., Naqvi, N.I., 2012. Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae. Autophagy 8, 1058e1070. Dou, X.Y., Wang, Q., Qi, Z.Q., Song, W.W., Wang, W., Guo, M., Zhang, H.F., Zhang, Z.G., Wang, P., Zheng, X.B., 2011. MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PloS One 6, e16439. Dufresne, M., Osbourn, A.E., 2001. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Mol. Plant Microbe Interact. 14, 300e307. Ebbole, D.J., 2007. Magnaporthe as a model for understanding host-pathogen interactions. Annu. Rev. Phytopathol. 45, 437e456. Gilbert, M.J., Thornton, C.R., Wakley, G.E., Talbot, N.J., 2006. A P-type ATPase required for rice blast disease and induction of host resistance. Nature 440, 535e539. Hoffman-Sommer, M., Kucharczyk, R., Piekarska, I., Kozlowska, E., Rytka, J., 2009. Mutations in the Saccharomyces cerevisiae vacuolar fusion proteins Ccz1, Mon1 and Ypt7 cause defects in cell cycle progression in a num1 Delta background. Eur. J. Cell. Biol. 88, 639e652. Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K., 2006. Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot. Cell 5, 1328e1336. Kucharczyk, R., Gromadka, R., Migdalski, A., Slonimski, P.P., Rytka, J., 1999. Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals. Yeast 15, 987e1000. Levine, B., Klionsky, D.J., 2004. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell. 6, 463e477. Liu, X.H., Lu, J.P., Zhang, L., Dong, B., Min, H., Lin, F.C., 2007. Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis. Eukaryot. Cell 6, 997e1005. Liu, X.H., Gao, H.M., Xu, F., Lu, J.P., Devenish, R.J., Lin, F.C., 2012. Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy 8, 1415e1425. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402e408. Lu, J.P., Liu, T.B., Lin, F.C., 2005. Identification of mature appressoriumenriched transcripts in Magnaporthe grisea, the rice blast fungus, using suppression subtractive hybridization. FEMS Microbiol. Lett. 245, 131e137.
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309 Mizushima, N., 2007. Autophagy: process and function. Genes Dev. 21, 2861e2873. Muren, E., Oyen, M., Barmark, G., Ronne, H., 2001. Identification of yeast deletion strains that are hypersensitive to brefeldin A or monensin, two drugs that affect intracellular transport. Yeast 18, 163e172. Nordmann, M., Cabrera, M., Perz, A., Brocker, C., Ostrowicz, C., Engelbrecht-Vandre, S., Ungermann, C., 2010. The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 Homolog Ypt7. Curr. Biol. 20, 1654e1659. Poteryaev, D., Spang, A., 2005. A role of SAND-family proteins in endocytosis. Biochem. Soc. Trans. 33, 606e608. Poteryaev, D., Fares, H., Bowerman, B., Spang, A., 2007. Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic. EMBO J. 26, 301e312. Rho, H.S., Kang, S., Lee, Y.H., 2001. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407e411. Sambrook, J., Fritsch, E.F., Maniatis, T., 2002. Molecular Cloning: a Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, New York, N.Y. Song, W., Dou, X., Qi, Z.Q., Wang, Q., Zhang, X., Zhang, H.F., Guo, M., Dong, S.M., Zhang, Z.G., Wang, P., Zheng, X.B., 2010. R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PloS One 5, e13193. Talbot, N.J., 2003. On the trail of a cereal killer: exploring the biology of Magnaporthe grisea. Annu. Rev. Microbiol. 57, 177e202. Talbot, N.J., Ebbole, D.J., Hamer, J.E., 1993. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575e1590.
309
Tartakoff, A.M., 1983. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell 32, 1026e1028. Tarutani, Y., Ohsumi, K., Arioka, M., Nakajima, H., Kitamoto, K., 2001. Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis. Gene 268, 23e30. Valent, B., 1990. Rice blast as a model system for plant pathology. Phytopathology 80, 33e36. Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N.,J., 2006. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312, 580e583. Wang, C.W., Stromhaug, P.E., Shima, J., Klionsky, D.J., 2002. The Ccz1Mon1 protein complex is required for the late step of multiple vacuole delivery pathways. J. Biol. Chem. 277, 47917e47927. Wang, C.W., Stromhaug, P.E., Kauffmann, E.J., Weisman, L.S., Klionsky, D.J., 2003. Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J. Cell. Biol. 163, 973e985. Weber, R.,W.,S., Wakley, G.,E., Thines, E., Talbot, N.,J., 2001. The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea. Protoplasma 216, 101e112. Wilson, R.A., Talbot, N.J., 2009. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nat. Rev. 7, 185e195. Xu, J.R., Staiger, C.J., Hamer, J.E., 1998. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc. Natl. Acad. Sci. U.S.A. 95, 12713e12718. Yi, M., Chi, M.H., Khang, C.H., Park, S.Y., Kang, S., Valent, B., Lee, Y.H., 2009. The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. Plant Cell 21, 681e695.
MoMon1 is required for vacuolar assembly, conidiogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae Hui-Min Gao a, Xiao-Guang Liu a, Huan-Bin Shi a, Jian-Ping Lu b, Jun Yang c, Fu-Cheng Lin a,c, Xiao-Hong Liu a,* a
State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou 310058, China b College of Life Sciences, Zhejiang University, Hangzhou 310058, China c China Tobacco Gene Research Center, Zhengzhou Tobacco Institute of CNTC, Zhengzhou 450001, China Received 29 February 2012; accepted 26 December 2012 Available online 29 January 2013
Abstract Mon1 protein is involved in cytoplasm-to-vacuole trafficking, vacuolar morphology and autophagy, and is required for homotypic vacuole fusion in Saccharomyces cerevisiae. Here we identify MoMON1 from Magnaporthe oryzae as an ortholog of S. cerevisiae MON1, essential for the morphology of the vacuole and vesicle fusion. Target gene deletion of MoMON1 resulted in accumulation of small punctuate vacuoles in the hypha and hypersensitivity to monensin, an antibiotic that blocks intracellular protein transport. The DMomon1 mutant exhibited significantly reduced aerial hyphal development and poor conidiation. Conidia of DMomon1 were able to differentiate appressoria. However, DMomon1 was non-pathogenic on rice leaves, even after wound inoculation. In addition, DMomon1 was slightly hypersensitive to Congo red and SDS, but not to cell wall degrading enzymes, suggesting significant alterations in its cell wall. The autophagy process was blocked in the DMomon1 mutant. Taken together, our results suggest that MoMON1 has an essential function in vacuolar assembly, autophagy, fungal development and pathogenicity in M. oryzae. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Conidiation; Vacuole; Autophagy; Virulence; Magnaporthe oryzae
1. Introduction The vacuole/lysosome is a terminal destination for ERmediated protein traffic in eukaryotic cells. Vesicle docking and fusion with a vacuole rely on a SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)-dependent reaction. As the first step, chaperones (NSF/Sec18p and -SNAP/Sec17p et al.) disassemble the CisSNARE complex (when on the same membrane) from the vacuole membrane; then, the C-Vps/HOPS (homotypic fusion and vacuolar protein sorting) complex is released from the SNAREs and reacts with rab GTPase Ypt7 (Ypt7p is required for all membrane fusion steps in which the vacuole is
* Corresponding author. Tel.: þ86 571 88982291; fax: þ86 571 88982183. E-mail address: [email protected] (X.-H. Liu).
involved) to accomplish tethering; finally, docking and fusion occur. The interaction of Mon1 and Ccz1 as a stable protein complex is required for the docking of SNARE complexes (Wang et al., 2003). The Mon1eCcz1 complex is not only essential for SNARE pair association during homotypic vacuole fusion, but also in other trafficking pathways to the vacuole (Wang et al., 2002). In yeast, the Mon1eCcz1 complex is regarded as a guanine nucleotide exchange factor (GEF) of Ypt7, which triggers endosomal maturation by activating Ypt7 in late endosomes (Nordmann et al., 2010). In yeast, MON1 was first identified as a gene involved in sensitivity to brefeldin A and monensin (Muren et al., 2001). Mon1 was classified into the SAND family despite its weak similarity with proteins from this family. MON1 orthologous genes were identified in major eukaryotes such as Fugu rubripes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana (Cottage et al., 2001; Hoffman-Sommer
0923-2508/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.resmic.2013.01.001
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
et al., 2009; Nordmann et al., 2010; Wang et al., 2002, 2003). Mon1 interacts with Ccz1 during fusion of vesicles and the vacuole membrane. Deletion of MON1 in yeast results in mutants with severe vacuolar protein-sorting defects. In C. elegans, MON1 orthologous gene SAND-1 is required for Rab7 function at the early and late endosomes and it regulates the cellular levels of both Rab5 and Rab7 (Poteryaev et al., 2007). Magnaporthe oryzae is a filamentous ascomycete fungus which is an excellent model organism for studying plantepathogen interactions (Talbot, 2003; Valent, 1990). The fungus infects its host through a specialized infection structure called appressorium that attaches tightly to the plant surface and directs penetration of the fungus into the host tissue by breaching the cuticle and plant cell wall (Ebbole, 2007; Talbot, 2003; Wilson and Talbot, 2009). Despite its importance, the M. oryzae ER to vacuole traffic is poorly understood. Recently, a P-type ATPase (APT2) was determined to be essential for exocytosis during plant infection (Gilbert et al., 2006). Two putative Hsp70 family proteins, Kar2 and Lhs1, were found to function as chaperones for protein translocation and maturation in the ER (Yi et al., 2009). The LHS1 gene is necessary for conidiogenesis and pathogenesis. MoSec22 and MoVam7, the orthologs of yeast SNARE proteins Sec22 and Vam7, play an important role in vacuolar morphogenesis, mycelial growth, sporulation, appressorium formation and pathogenicity (Dou et al., 2011; Song et al., 2010). MGG_12832 protein shares overlapping functions with Snx41 and Atg20. MoSnx41 associates with autophagosomes and is required for pathogenicity. The Snx41-GFP is present as puncta or short tubules that partially associate with autophagosomes or are distributed on the surface of the vacuoles during vegetative and pathogenic development in M. oryzae (Deng et al., 2012). Despite accumulated knowledge of the yeast Mon1, little is known about Mon1 proteins from filamentous fungi. In this study, we investigated the possible functions of MoMON1 in M. oryzae and showed that its deletion led to important defects in aerial hyphal development, sporulation, autophagy, appressoria formation and plant pathogenicity. 2. Materials and methods 2.1. Fungal strains and culture conditions The wild-type strain Guy11 and all mutants described in this study were maintained and cultured on complete medium plates (CM) at 25 C with standard growth and storage procedures (Talbot et al., 1993). MM media (6 g NaNO3, 0.52 g KCl, 0.52 g MgSO4, 1.52 g KH2PO4, 10 g glucose, 0.5% biotin) was used to characterize growth of the strains. To observe sexual reproduction, strains were cultured on oatmeal agar medium (OMA) (30 g oatmeal in 1 l distilled water) for 4 weeks. 2.2. DNA/RNA manipulation and quantitative RT-PCR Most general molecular biology techniques for nucleic acid analysis, including cloning, restriction digest, gel electrophoresis, ligation reaction and DNA gel blot hybridization
301
analysis were performed according to standard protocols (Sambrook et al., 2002). For genomic DNA isolation and RNA extraction, cultures were grown in liquid CM at 25 C for 3e4 days with 150-rpm shaking. For quantitative RT-PCR, mycelia were collected and subjected to ER stress treatments, such as DTT (10 mM) for 30 min and tunicamycin (10 mg/mL) for 1 h. The mycelia were collected and quickly frozen in liquid N2 for RNA extraction. Total RNA of the above mixture was isolated by a Trizol method following the manufacturer’s procedure (Molecular Research Center, Inc., USA). cDNA reverse transcribed from 500 ng of total RNA with random 6 mers using the SYBRÒ ExScriptTM RT-PCR kit (TaKaRa Bio Co Ltd, Dalian, China) was diluted to 1:3 with nuclease-free water. Real-time PCR reactions were performed in 20 ml volume containing 200 nM of each primer, 0.5 ml of cDNA and 10 ml of 2 SYBRÒ Premix Ex Taq (TaKaRa Bio Co Ltd, Dalian, China). Real-time PCR was run on a MastercyclerÒ ep realplex (Eppendorf, USA). The cycling conditions were: 30 s at 95 C followed by 40 cycles of 5 s at 95 C and 31 s at 60 C. Fluorescence signal data were collected during the 60 C phase of each cycle. Melt curves from 72 C to 60 C in 0.5 C increments, measuring fluorescence at each temperature, were collected for all samples following the last cycle. To analyze relative abundance of transcripts with the 2DDCt method (Livak and Schmittgen, 2001), the average threshold cycle (Ct) was normalized by b-tubulin for each condition as 2DCt. Fold-changes were calculated as 2DDCt. The primer pairs used for quantitative RT-PCR are listed in Supplemental Table 1. The PCR reactions were repeated at least twice independently with three replicates, and a representative set of data was presented. 2.3. Isolation and sequence analysis of MoMON1 A full-length cDNA fragment of MoMON1 was isolated from the cDNA library of Guy11 (Lu et al., 2005) using primers Mp1 and Mp2; PCR primer pairs were designed based on the M. oryzae genome database (http://www.broadinstitute. org/annotation/genome/magnaporthe_comparative/ MultiHome.html). cDNA of MoMON1 was then cloned into the pUCm-T T-vector (Sangon, Shanghai, China) and verified by sequencing. The ClustalW program was used for amino acid sequence alignments and the calculated phylogenetic tree was established using MEGA 4.0 Beta program. 2.4. Disruption of MoMON1 and complementation of the DMomon1 mutant To replace the gene, two flanking sequences of MoMON1 were amplified from the Guy11 genomic DNA and inserted into the pBS-HPH1 vector (Liu et al., 2007). A 1.02-kb upstream of MoMON1 was amplified with primers Mupp1 and Mupp2 and cloned between the XhoI and SalI sites on the pBS-HPH1 vector to generate pBS-HPH1-up. Then, a 1.01 kb downstream of MoMON1 was amplified with primers Mdwp1 and Mdwp2 and inserted into the PstI and XbaI sites of pBS-HPH1-up to
302
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
generate the pBS-MoMon1 vector. Finally, the specific fragment containing the MoMON1 upstream fragment, hygromycin gene and downstream fragment were dissected with XhoI/XbaI from the pBS-MoMon1 vector inserted into the corresponding sites of pCAMBIA1300 to generate the final deletion vector p1300-MON1. The replacement vector was introduced into Agrobacterium tumefaciens strain AGL-1 and then transformed into M. oryzae Guy11 by the ATMT method to generate MoMON1 null mutants, as described previously (Rho et al., 2001). Candidate transformants were selected by 200 ug/ml hygromycin B and were checked by PCR using primers Myzp1 and Myzp2. Then, genomic DNA of candidate transformants was extracted under standard conditions. Southern blot were carried out according to the protocol of the digoxigenin (DIG) high prime DNA labeling and detection starter kit I (Roche, Germany). For Southern hybridization analysis, the genomic DNA was digested with ApaI and separated in a 0.7% agarose gel. The labeled probe was amplified from genomic DNA of Guy11 using primers Mdwp1 and Mdwp2. For complementation assays, the 4.38-kb PCR product contained a 1.5-kb upstream sequence, a full-length MoMON1 gene coding region and a 0.98-kb downstream sequence, was amplified from the Guy11 genomic DNA using primers Mcop1 and Mcop2 and cloned into the pGEM-T easy vector (Promega, UK). The EcoRI/XbaII-digested PCR fragment was then ligated to the EcoRI/XbaI site in pCAMBIA1300X-SUR to obtain p1300SUR-MON1; the selectable marker SUR gene (sulfonylurea-resistant gene) was obtained from the pCB1532 vector and cloned into the EcoRV/EcoRI site of pCAMBIA1300X. The AGL-1 strain with the complementation vector was transformed using ATMT (A. tumefaciens-mediated transformation), by co-culture with the DMoMON1 strain. Transformants were selected on DCM (yeast nitrogen base without amino acid 1.7 g/l, NH4NO3 2 g/l, L-asparagine 1 g/l, glucose 10 g/l, Na2HPO4 used to adjust pH to 6.0) supplemented with 100 mg/mL sulfonylurea. 2.5. Phenotypic analysis For conidia production, strains were grown on CM medium at 25 C with a 12-h photophase for 7 days. Conidia were harvested from three mycelial discs (1 cm in diameter) and suspended in 3 ml sterile distilled water by vortexing and counted with a hemocytometer under a microscope. For growth, mycelial plugs (5 mm in diameter) were transferred from 7-day-old CM plates and grown on other fresh medium (OMA, CM with agents), then incubated at 25 C with a 12-h photophase. The radial growth of vegetative mycelia was measured after 4 days and 8 days. Conidial germination and appressorium formation were measured on plastic cover slips at a concentration of 2 104 conidia/ml in sterile distilled water and incubated at 25 C for 2e48 h. Conidial germination and appressoria formation were observed and counted under the microscope. ID50 values (effective concentration to inhibit fungal growth by 50%) were determined for each compound by calculating the percent inhibition [(mean colony diameter on
unamended media mean colony diameter on amended media)Omean colony diameter on media] and subjecting data to probit analysis software. All data were valid with P < 0.05 and efficient >95%.
compoundunamended using DPS relative co-
2.6. Pathogenicity assay Pathogenicity assays were performed as described previously (Liu et al., 2007). For spray infection assays, the suspension with 2 104 conidia/ml was sprayed wellproportioned onto plant leaves using an artist’s airbrush (Badger Co., IL); 2-week-old seedlings of rice (Oryza sativa cv. CO-39) were used. Inoculated rice were placed in a dew and dark chamber at 22 C for 48 h and then transferred to the growth chamber with a photoperiod of 12 h. Lesion formation was examined after 7-day incubation. For the cut leaf assay, mycelial plugs (10-day-old, 1 cm in diameter) were removed from CM medium and deposited onto the upper surface of the isolated barley cut leaves, and then incubated at 25 C with a 12-h photophase for 4 days; 8-dayold barley (Hordeum vulgare cv. ZJ-8) seedlings were used. The root infection assay was carried out as described previously (Dufresne and Osbourn, 2001). 2.7. Electron microscopy Conidia collected from 10-day-old mycelia were cultured at 28 C for 48 h in CM liquid medium with continuously shaking at 150 rpm. The mycelia were collected, washed in sterile distilled water to the full, then transferred to distilled water with 2 mM phenylmethylsulfonyl fluoride (PMSF) and incubated at 28 C for 4 h with shaking at 150 rpm. The mycelia cultured in CM liquid medium for 48 h and PMSFinduced mycelia were collected respectively, and the samples were fixed and postfixed as described previously (Liu et al., 2007). After that, the samples were examined under a JEM-1230 electron microscope (JEOL, Tokyo, Japan) operating at 70 kV. 3. Results 3.1. Identification and deletion of the MoMON1 gene in M. oryzae The protein sequence of yeast Mon1 was used to search the M. oryzae genome database using BlastP, identifying MGG_05755.7 as the most similar protein (30% identity). MoMON1 was designated. Then the full coding sequence (CDS) was cloned from Guy11 using high-fidelity PCR and subsequently sequenced (GenBank no.: JN845639). The CDS obtained is 1893 bp in length and putatively encodes a protein of 630 amino acids. The MoMon1 protein sequence from Guy11 was identical to MoMon1 from 70 to 15 (MGG_05755.7) and displayed a high level of similarity to Mon1 from other fungi, such as Aspergillus oryzae (XP_001819506.2, 91% identity), Gibberella zeae
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
303
(XP_387259.1, 96% identity) and Trichoderma reesei (EGR50254.1, 93% identify). A phylogenetic tree of Mon1related proteins identified in different fungal genomes is shown in Fig. 1. To determine the molecular function of MoMon1 in M. oryzae, we constructed a deletion mutant by targeted gene replacement using ATMT (Fig. S2A). Southern blot analyses were performed to confirm single-copy genomic integration and exclude additional ectopic integrations. An approximately 7.5 kb band was detected in mutants m-12 and m-35, in contrast to an approximately 3.9 kb band in wild type Guy11 (Fig. S2B). Two mutants (M-12 and M-35) showed comparable phenotypes and one (M12) was chosen for further studies. Complementation assay was carried out and transformant Com-12, which contained a full-length gene copy of MoMON1 (data not shown), was selected for further studies.
plates. Some conidia exhibited abnormal morphologies compared to wild type or Com-12 (Fig. 2C). Conidiophores and conidial formation were further analyzed. Small amounts of hyphae, conidiophores and conidia were observed in the DMomon1 mutant at 24 h post-conidial induction after 8-day incubation (Fig. 2D). Conidial germination and appressorium formation rates of the DMomon1 mutant were assessed. The conidial germination rate of the mutant was similar to the wild type, but only 50% of these conidia differentiated appressoria, compared to 99% in the wild type. Our results showed that MoMON1 is required for full mycelial growth, sporulation and appressorium formation.
3.2. MoMON1 affects vegetative growth, conidiogenesis and appressorium formation
Because the mycelial growth of the MoMON1 mutant was interrupted, we further monitored the effects of various cellwall-perturbing agents on the DMomon1 mutant. Mycelial growth was measured on CM plates amended with compounds (SDS, Congo red and caffeine) that cause cell wall stress, respectively (Choi et al., 2009). Mycelial growth of the DMomon1 mutant and wild-type strain was reduced on CM containing Congo red or SDS, and the DMomon1 mutant displayed sensitivity to Congo red and SDS. Mycelial growth of the DMomon1 mutant was reduced on CM containing Congo red or SDS compared to the wild type (Fig. 3). The wild type ID50 value for SDS was similar to the value for the DMomon1 mutant, but the wild type ID90 value for SDS was 2.3-fold higher than that for the DMomon1mutant (Table 1). The ID50 value of Congo red for the wild type was about 2.5fold higher than that of the DMomon1 mutant (Table 1). Calcofluor white (CFW) was used to probe cell wall integrity. It was mostly distributed at the septa and tips where chitin, one of the main components of the fungal cell wall, was actively synthesized. There was no significant difference between Guy11, the mutant and Com-12 (Fig. S4). The enzymatic activity of glucanex was assayed using culture filtrates from the wild type and the mutant grown in CM liquid media. No significant difference in the rate of protoplast release was found at 30 min (Fig. S5). It is reported that caffeine has an effect on a variety of processes, including changes in cell morphology in yeast (Kucharczyk et al., 1999); we further tested the effect of caffeine in M. oryzae. As illustrated in Fig. 3, the DMomon1 mutant was only slightly more sensitive to caffeine than the wild type (wild type ID50 1.3-fold higher than DMomon1 value, Table 1). Since Mon1 in yeast was initially identified during screening of deletion strains sensitive to monensin, which interferes with intracellular trafficking, we tested the sensitivity of the DMomon1 mutant to monensin in M. oryzae. The DMomon1 mutant colonies were unable to extend on CM media with 5 ug/ml monensin, while the wild-type strain colonies continued to slowly grow (Fig. 3), suggesting that deletion of MoMON1 leads to hypersensitivity to monensin. The ID50 value of monensin for Guy11 was about 2-fold higher than that of the DMomon1 mutant (Table 1). The findings above indicate that deletion of
The morphology of the strains was monitored in different media. The DMomon1 mutant showed reduced vegetative growth in comparison to wild-type strain Guy11 and rescued strain Com-12 on CM, OMA and MM medium (Fig. 2A). The DMomon1 mutant produced sparse aerial hyphae, in contrast with the dense aerial hyphae of the wild-type strain and rescued strain Com-12; especially in OMA and MM medium plates, the DMomon1 mutant lacked aerial hyphae (Fig. 2A). Deletion of MoMON1 had a serious effect on mycelial growth, with slight decrease in mature hyphae (Fig. S3). We found that the DMomon1 mutant scarcely produced spores (w0.5%) relative to the wild-type strain on CM plates (Fig. 2B) and no spore was produced on OMA or MM medium
Fig. 1. Phylogenetic tree constructed using MEGA4.0 beta program neighborjoining tree with 1000 bootstrap replicates of phylogenetic relationships between Mon1 homologs in eukaryotes. The compared sequences were from M. oryzae (MoMon1), S. cerevisiae (CAA96832.1), N. crassa (XP_959164.2), M. musculus (NP_766603.1), H. sapiens (NP_115731.2), D. melanogaster (NP_608868.1), Gibberella zeae (XP_387259.1), Trichoderma reesei (EGR50254.1), Neurospora tetrasperma (EGO52340.1), Fusarium oxysporum (EGU75269.1), Aspergillus clavatus (XP_001272699.1), Phaeosphaeria nodorum (XP_001802326.1) and Chaetomium globosum (XP_001220171.1). The numbers at branch nodes are bootstrap values.
3.3. MoMON1 is hypersensitive to vesicular transport inhibitors and displays cell wall defects
304
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
Fig. 2. Growth and sporulation of the DMomon1 mutant. (A) Guy11, the DMomon1 mutant, and DMomon1 complement strain Com-12 were grown on CM, OMA and MM medium for 8 days. The DMomon1 mutant displayed extremely sparse aerial hyphae, especially on MM medium plates. (B) Few conidia were collected in the DMomon1 mutant, in contrast to Guy11 and Com-12. Error bars represent standard deviations. Reduction is significant (P ¼ 0.01) according to Duncan’s multiple range test. (C) Morphology of the conidia. Conidia were collected from colonies of strains on CM media. The DMomon1 mutant showed abnormal conidia. Scale bar ¼ 10 mm. (D) Development of conidia on conidiophores was observed under a light microscope 24 h after induction of conidiation under cover slips. Few conidia and conidiophores developed in the DMomon1 mutant. Scale bar ¼ 50 mm.
the MoMON1 in M. oryzae altered cell wall integrity and led to higher sensitivity to cell wall disturbing agents and vesicular transport inhibitors. 3.4. MoMON1 is involved in the protein vesicular traffic To characterize whether expression of vacuole trafficking and ER-related genes such as PDE1, MgAPT2, LHS1, KAR2, MoVAM7, MoSEC22 and MoSNX41 was affected by the MoMON1 mutation, quantitative RT-PCR was performed
using RNA extracted from cultures of the wild type and the mutant after treatment with inhibitors. Cultures of the wild type and mutant were treated with 10 mM DTT (ER stress inducer) or 10 mg/mL tunicamycin (protein glycosylation and traffic inhibitor). Expression data on marker genes were assessed under these conditions in the DMomon1 mutant compared to the wild type (Fig. 4). Upon DTT treatment, the expression levels of MoVAM7, MoSEC22, LHS1 and KAR2 were strongly decreased compared to their upregulation in the wild type (Fig. 4A). This was also observed for MoSEC22,
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
305
vacuole. In the DMomon1 mutant, neutral red staining was localized in small vacuoles, while it stained large globular vacuoles in the wild type (Fig. 5A). FM4-64 staining confirmed that the DMomon1 mutant accumulated numerous small vesicles (Fig. 5B). Ultrastructural analysis using transmission electron microscopy showed that the hyphae of DMomon1 accumulated a large number of vesicles, while typical large vacuoles were observed in Guy11 and Com-12 (Fig. 5C). Based on previous reports, fusion of autophagosomes with the vacuole and subsequent breakdown of the singlemembrane autophagic body in the vacuole is necessary for the complete autophagy pathway (Levine and Klionsky, 2004). However, our results showed that, when cultured in sterile distilled water (starvation-induce) in the presence of 2 mM PMSF for 4 h, no autophagic bodies were observed in the lumen of vacuoles in DMomon1 mutant hyphal cells (Fig. 5C). Thus, we concluded that the autophagy pathway was blocked
Fig. 3. Effects of cell-wall-disorganizing agents upon the DMomon1 mutant. The DMomon1 mutant, Guy11 and Com-12 were incubated on CM plates supplemented with various stress inducers at 28 C for 8 days. Growth of the strains in media supplemented with SDS (0.01%), Congo Red (200 mg/ml), caffeine (2 mM), and monensin (5 ug/ml).
LHS1 and KAR2 upon tunicamycin treatment (Fig. 4B). These results suggest that the absence of MoMON1 impairs the transcriptional response induced by ER stress.
3.5. MoMON1 is required for the autophagy pathway Cytological analyses showed that DMomon1 accumulated numerous vesicles that might be small vacuoles. Vital dyes neutral red and FM4-64 were used to stain vesicles of the wild type and the mutant. In normal cells of Guy11 or Com-12, neutral red was quickly internalized, enabling staining of the
Table 1 ID50 and ID90 values for cell-wall-perturbing agents. Strains ID50 ID90
Guy11 DMomon1 Guy11 DMomon1
Congo red (mg/ml)
SDS (%)
Caffeine (mM)
Monensin (ug/ml)
0.327 0.138 4.930 3.411
0.010 0.008 0.037 0.016
3.742 2.927 8.057 5.433
0.310 0.165 30.932 8.185
Fig. 4. Effect of DTT and tunicamycin on expression of ER-related genes in the wild type and DMomon1 mutant. Mycelia were grown in lipid complete medium for 2 days and then subjected to treatment with 10 mM DTT (A) or 10 mg/mL tunicamycin (B). Expression data were normalized using the btubulin gene and calibrated against the profile of mycelia grown without treatment. Error bars represent standard deviations and asterisks represent significant differences among stains tested. Reductions were significant (P ¼ 0.01 or P ¼ 0.05) according to Duncan’s multiple range test.
306
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
in the DMomon1 mutant under starvation conditions in M. oryzae. 3.6. MoMON1 plays a key role in pathogenicity Pathogenicity assays of the DMomon1 mutant were performed on two susceptible hosts (rice and barley). Since the DMomon1 mutant produced few conidia, we used weak inocula (2 104 conidia/ml) for spray inoculation. Results showed that the wild-type strain and the MoMON1 complementary strain caused visible brown and punctuated lesions on rice leaves, whereas the DMomon1 mutant caused no macroscopic lesions (Fig. 6A). Mycelial plugs of DMomon1 inoculated onto detached barley did not cause disease symptoms, while the wild-type strain induced susceptible lesions. Although the cuticle of detached barley was damaged, there were no visible lesions when mycelial plugs of DMomon1 were inoculated (Fig. 6B). In the root infection assay, no visible brown lesions developed on rice roots when the DMomon1 mutant mycelial plugs were inoculated (Fig. 6C). In contrast, the wild-type strain and the MoMON1 complement strain caused typical rice blast lesions in the same tissues after 3 weeks of inoculation. Taken together, these data suggested that MoMON1 is indispensable for the pathogenicity of M. oryzae. 4. Discussion In yeast, Mon1 is a monensin hypersensitivity protein required for nearly all ER traffic pathways, as well as in ER for the vacuole protein sorting pathway (Wang et al., 2002, 2003). We isolated MoMON1, an M. oryzae ortholog of yeast MON1. Targeted deletion of MoMON1 has a strong effect on sporulation, vacuolar morphology, autophagy and pathogenicity in rice and barley. Conidiation is one of the key stages of the rice blast fungus for carrying out the infectious cycle. The DMomon1 mutant is defective in aerial hypha formation and conidiogenesis, and most of the conidia are abnormal. MPS1 is a mitogen-activated protein-kinase-encoding gene responsible for regulation of cell wall growth under membrane stress conditions (Xu et al., 1998). Similarly to the DMomon1 mutant, the Dmps1 mutant also showed dramatically reduced aerial hypha formation and conidiation (Xu et al., 1998). However, progressive autolysis was observed in the colony of the Dmps1 mutant in contrast to the sparse aerial hypha of the DMomon1 mutant.
Fig. 5. Fragmented vacuole in the hyphae of the DMomon1 mutant. (A) Neutral red staining of vacuoles. Numerous small vacuoles are observed in theDMomon1 mutant, while large globular vacuoles are observed in the wild type and Com12. Scale bar ¼ 10 mm (B) FM4-64 staining of membranes of neutral vacuoles in Guy11 and Com-12. Small vacuoles were found in the
DMomon1 mutant. Strains were grown for 24 h on CM-overlaid microscope slides before staining. Scale bar ¼ 10 mm. (C) Blocking of vacuole-related fusion pathways in the DMomon1 mutant. Left side: mycelia cultured at 28 C for 48 h in CM liquid medium continuously shaken at 150 rpm. Right side: starvation-induced mycelia, where autophagy pathways were triggered. In the DMomon1 mutant, numerous smaller vacuoles were presented under nutrient and starvation conditions; the organelle and autophagosomes were detected in vacuoles of mycelia PMSF-induced from Guy11 and Com-12 (indicated by the arrow), whereas no autophagic body was evident in DMomon1 mutant vacuoles.
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
307
Fig. 6. Loss of pathogenicity of the DMomon1 mutant. (A) Rice seedlings aged two weeks were inoculated by spraying 2 104 conidia/ml of Guy11, DMomon1 mutant and Com-12 individually with 0.2% gelatin as control. After 7-day inoculation, lesion formation on rice leaves was observed. (B) Disease symptoms on cut leaves of barley (intact and wounded) inoculated with mycelial plugs from Guy11 and the DMomon1 mutant and Com-12, individually with agar as control. Typical leaves were photographed 4 days after inoculation. (C) Root infection assay. Arrows show necrotic lesions, the DMomon1 mutant, Guy11, undisposed rice as control and Com-12.
MoSEC22 and MoSNX41 are required for sporulation in M. oryzae (Deng et al., 2012; Song et al., 2010). The DMomon1 mutant has a defect in conidiation, but the few conidia were able to develop appressoria at the tip of the germ tube. Unlike the DMovam7 mutant, conidia could not differentiate appressoria (Dou et al., 2011). Taken together, we reasoned that MoMON1 plays a role in formation of conidia and appressoria. Fungal cell walls have a complex structure, and many stresses or developmental processes, as well as plant infection, can induce their remodeling (Cabib et al., 2001). Our results showed that mycelial growth of the DMomon1 mutant decreased in the presence of caffeine, SDS and Congo red. These observations are in accordance with earlier reports on
MoVam7 and Mosec22 (Dou et al., 2011; Song et al., 2010). The DMomon1 mutant was found to be insensitive to cellwall-digesting enzymes. Unlike Dmps1, it produced protoplasts at a faster rate than Guy11 under treatment of cellwall-digesting enzymes (Xu et al., 1998). Interestingly, Dmosec22 was resistant to lysing enzymes (Song et al., 2010). These results indicate that deletion of MoMON1 influenced the composition and/or structure of the cell wall, and the resulting mutant showed differences from those with deletion of other genes, such as MPS1 and MoSEC22. qRT-PCR analyses showed that genes normally upregulated in response to ER stress (MoSEC22, LHS1, KAR2, MoVAM7 Yi et al., 2009) were downregulated in theDMomon1 mutant
308
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309
compared to the wild type. These results suggest that MoMON1 is required for the transcriptional response induced by ER stress. The hypersensitivity of the DMomon1 mutant to monensin, an inhibitor of vesicle trafficking in eukaryotes (Tartakoff, 1983), strongly suggests that this mutant is altered in intracellular protein trafficking. In yeast, the Mon1 protein forms a stable complex with the Ccz1 protein which binds to the vacuole membrane. The Ccz1eMon1 complex is known to function at the tethering/ docking stage of vacuole membrane fusion or homotypic vacuole fusion, and deletion of either Ccz1 or Mon1 also resulted in a fragmented vacuole phenotype in Saccharomyces cerevisiae (Wang et al., 2002, 2003; Weber et al., 2001). Likewise, the Mon1 ortholog SAND-1 is required for late steps of endocytosis in C. elegans. The sand-1 (or552) mutants displayed abnormal receptor-mediated endocytosis (Poteryaev and Spang, 2005; Poteryaev et al., 2007). Additionally, in Aspergillus nidulans, disruption of the VpsA gene essential for vacuolar biogenesis leads to fragmented vacuoles (Tarutani et al., 2001); in M. oryzae, null mutants of the SNAREencoding genes MoVAM7 and MoSEC22 also displayed small fragmented vacuoles (Dou et al., 2011; Song et al., 2010). Furthermore, cytological analyses revealed that numerous small vacuoles accumulated in the DMomon1 mutant. These results suggest that MoMON1 is required for fusion of vesicles and vacuoles, as observed in yeast. It has been reported that the autophagy pathway is a common and evolutionarily conservative process that delivers proteins and organelles to the vacuole (Mizushima, 2007). The autophagy pathway that delivers organelles and cytoplasmic content to the vacuole has been shown to be essential for fungal development and pathogenicity (Bartoszewska and Kiel, 2012; Liu et al., 2012). We showed that hypha of the DMomon1 mutant displayed fragmented vacuoles and that the autophagic pathway was blocked under either nutrient or starvation conditions. Similarly, the autophagic gene MoATG1 resulted in a reduction in appressorium turgor and loss of pathogenicity (Liu et al., 2007). The MgATG8 null mutant is also impaired in autophagy and pathogenicity (VeneaultFourrey et al., 2006). In A. oryzae, the DAoatg8 mutant also had defects in the formation of conidia and aerial hyphae (Kikuma et al., 2006). However, the morphology of the vacuole was not altered in DMgatg1, DMgatg8 and DAoatg8 mutants. Taken together, our studies suggest that autophagy was blocked in the DMomon1 mutant and resulted from defects in vacuole-related fusion pathways. In summary, we have identified and characterized MoMON1, a gene encoding a vacuolar fusion protein in M. oryzae. MoMON1 plays an important role in the differentiation processes of M. oryzae, including intracellular transport and development in vegetative growth, conidiogenesis, appressorium formation and pathogenicity toward plants. Acknowledgments We thank the anonymous reviewers for their careful and thoughtful reviews and helpful advice.
This work was supported by grants (No. 30925029 and 31000077) funded by the National Natural Science Foundation of China. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.resmic.2013.01.001. References Bartoszewska, M., Kiel, J.A., 2012. The role of macroautophagy in development of filamentous fungi. Antioxid. Redox Signal 14, 2271e2287. Cabib, E., Roh, D.H., Schmidt, M., Crotti, L.B., Varma, A., 2001. The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J. Biol. Chem. 276, 19679e19682. Choi, J., Kim, Y., Kim, S., Park, J., Lee, Y.H., 2009. MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae. Fungal Genet. Biol. 46, 243e254. Cottage, A., Edwards, Y.J.K., Elgar, G., 2001. SAND, a new protein family: from nucleic acid to protein structure and function prediction. Comp. Funct. Genom. 2, 226e235. Deng, Y.Z., Qu, Z., He, Y., Naqvi, N.I., 2012. Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae. Autophagy 8, 1058e1070. Dou, X.Y., Wang, Q., Qi, Z.Q., Song, W.W., Wang, W., Guo, M., Zhang, H.F., Zhang, Z.G., Wang, P., Zheng, X.B., 2011. MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PloS One 6, e16439. Dufresne, M., Osbourn, A.E., 2001. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Mol. Plant Microbe Interact. 14, 300e307. Ebbole, D.J., 2007. Magnaporthe as a model for understanding host-pathogen interactions. Annu. Rev. Phytopathol. 45, 437e456. Gilbert, M.J., Thornton, C.R., Wakley, G.E., Talbot, N.J., 2006. A P-type ATPase required for rice blast disease and induction of host resistance. Nature 440, 535e539. Hoffman-Sommer, M., Kucharczyk, R., Piekarska, I., Kozlowska, E., Rytka, J., 2009. Mutations in the Saccharomyces cerevisiae vacuolar fusion proteins Ccz1, Mon1 and Ypt7 cause defects in cell cycle progression in a num1 Delta background. Eur. J. Cell. Biol. 88, 639e652. Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K., 2006. Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot. Cell 5, 1328e1336. Kucharczyk, R., Gromadka, R., Migdalski, A., Slonimski, P.P., Rytka, J., 1999. Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals. Yeast 15, 987e1000. Levine, B., Klionsky, D.J., 2004. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell. 6, 463e477. Liu, X.H., Lu, J.P., Zhang, L., Dong, B., Min, H., Lin, F.C., 2007. Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis. Eukaryot. Cell 6, 997e1005. Liu, X.H., Gao, H.M., Xu, F., Lu, J.P., Devenish, R.J., Lin, F.C., 2012. Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy 8, 1415e1425. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402e408. Lu, J.P., Liu, T.B., Lin, F.C., 2005. Identification of mature appressoriumenriched transcripts in Magnaporthe grisea, the rice blast fungus, using suppression subtractive hybridization. FEMS Microbiol. Lett. 245, 131e137.
H.-M. Gao et al. / Research in Microbiology 164 (2013) 300e309 Mizushima, N., 2007. Autophagy: process and function. Genes Dev. 21, 2861e2873. Muren, E., Oyen, M., Barmark, G., Ronne, H., 2001. Identification of yeast deletion strains that are hypersensitive to brefeldin A or monensin, two drugs that affect intracellular transport. Yeast 18, 163e172. Nordmann, M., Cabrera, M., Perz, A., Brocker, C., Ostrowicz, C., Engelbrecht-Vandre, S., Ungermann, C., 2010. The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 Homolog Ypt7. Curr. Biol. 20, 1654e1659. Poteryaev, D., Spang, A., 2005. A role of SAND-family proteins in endocytosis. Biochem. Soc. Trans. 33, 606e608. Poteryaev, D., Fares, H., Bowerman, B., Spang, A., 2007. Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic. EMBO J. 26, 301e312. Rho, H.S., Kang, S., Lee, Y.H., 2001. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407e411. Sambrook, J., Fritsch, E.F., Maniatis, T., 2002. Molecular Cloning: a Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, New York, N.Y. Song, W., Dou, X., Qi, Z.Q., Wang, Q., Zhang, X., Zhang, H.F., Guo, M., Dong, S.M., Zhang, Z.G., Wang, P., Zheng, X.B., 2010. R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PloS One 5, e13193. Talbot, N.J., 2003. On the trail of a cereal killer: exploring the biology of Magnaporthe grisea. Annu. Rev. Microbiol. 57, 177e202. Talbot, N.J., Ebbole, D.J., Hamer, J.E., 1993. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575e1590.
309
Tartakoff, A.M., 1983. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell 32, 1026e1028. Tarutani, Y., Ohsumi, K., Arioka, M., Nakajima, H., Kitamoto, K., 2001. Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis. Gene 268, 23e30. Valent, B., 1990. Rice blast as a model system for plant pathology. Phytopathology 80, 33e36. Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N.,J., 2006. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312, 580e583. Wang, C.W., Stromhaug, P.E., Shima, J., Klionsky, D.J., 2002. The Ccz1Mon1 protein complex is required for the late step of multiple vacuole delivery pathways. J. Biol. Chem. 277, 47917e47927. Wang, C.W., Stromhaug, P.E., Kauffmann, E.J., Weisman, L.S., Klionsky, D.J., 2003. Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J. Cell. Biol. 163, 973e985. Weber, R.,W.,S., Wakley, G.,E., Thines, E., Talbot, N.,J., 2001. The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea. Protoplasma 216, 101e112. Wilson, R.A., Talbot, N.J., 2009. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nat. Rev. 7, 185e195. Xu, J.R., Staiger, C.J., Hamer, J.E., 1998. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc. Natl. Acad. Sci. U.S.A. 95, 12713e12718. Yi, M., Chi, M.H., Khang, C.H., Park, S.Y., Kang, S., Valent, B., Lee, Y.H., 2009. The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. Plant Cell 21, 681e695.