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American Society of Plant Biologists Peroxisomal Metabolic Function Is Required for Appressorium-Mediated Plant Infection by Colletotrichum lagenariumLaboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan 1 To whom correspondence should be addressed. E-mail ytakano{at}kais.kyoto-u.ac.jp; fax 81-75-753-6131
Peroxisomes are organelles that perform a wide range of metabolic functions in eukaryotic cells. However, their role in fungal pathogenesis is poorly understood. Here we report that ClaPEX6, an ortholog of PEX6, is required for the fungus Colletotrichum lagenarium to infect host plants. ClaPEX6 was identified in random insertional mutagenesis experiments aimed at elucidating genes involved in pathogenesis. Functional analysis, using a green fluorescent protein cassette containing the peroxisomal targeting signal1 (PTS1), revealed that import of PTS1-containing proteins is impaired in clapex6 mutants generated by targeted gene disruption. Failure of growth on fatty acids shows an inability of fatty acid  -oxidation in these mutants. These results indicate that disruption of ClaPEX6 impairs peroxisomal metabolism, even though clapex6 mutants show normal growth and conidiation in nutrient-rich conditions. The clapex6 mutants formed small appressoria with severely reduced melanization that failed to form infectious hyphae. These data indicate that peroxisomes are necessary for appressorium-mediated penetration of host plants. The addition of glucose increased the pathogenicity of clapex6 mutants, suggesting that the glucose metabolic pathway can compensate partially for peroxisomes in phytopathogenicity.
Peroxisomes are single-membrane-bound organelles possessing multiple metabolic functions, including -oxidation of fatty acids, glyoxylate metabolism, and metabolism of reactive oxygen species. They also are required for specific functions such as methanol assimilation in some yeast and penicillin biosynthesis in the filamentous fungus Penicillium chrysogenum (Müller et al., 1991 ; van den Bosch et al., 1992; Subramani, 1993). In plants, peroxisomes are known to differentiate into at least three different classes: glyoxysomes, leaf peroxisomes, and unspecialized peroxisomes (Beevers, 1979), and it has been shown that leaf peroxisomes contain some of the enzymes involved in photorespiration (Tolbert, 1982). Most peroxisomal matrix enzymes have been shown to contain one of two peroxisome targeting signals (PTSs) (Subramani, 1993). Extensive analysis of yeast mutants has identified >20 PEX genes whose products, peroxins, are required for peroxisome biogenesis (van der Klei and Veenhuis, 1997; Subramani, 1998). In humans, peroxisome biogenesis disorders, such as Zellweger syndrome, are a group of lethal inherited diseases (Lazarow and Moser, 1989). Human orthologs of the yeast PEX genes have been characterized as causative genes for peroxisome biogenesis disorders (Gould and Valle, 2000). Compared with studies of PEX genes in mammals and yeast, functional analysis of PEX genes in other organisms is limited to a few reports (Berteaux-Lecellier et al., 1995; Lin et al., 1999; Hayashi et al., 2000). There are no reports regarding the roles of PEX genes for fungal pathogens. Genetic and physiological studies have provided an increasing understanding of fungal infection mechanisms; however, it remains unclear whether peroxisomal functions are prerequisite for fungal phytopathogenicity.
Plant pathogenic fungi produce asexual spores called conidia for their reproduction. Conidia formed in lesions of infected plants are dispersed by wind or water splash. Once conidia land on aerial parts of the host plants, they start morphological development and secretion of the low-molecular-weight or enzymatic compounds required for infection. On the surface of host plants, conidia are exposed to limited nutrient conditions and require the use of storage compounds, such as lipids and carbohydrates, to infect their host plant. Many phytopathogens, including Colletotrichum species, which cause destructive anthracnose diseases on numerous crops and ornamental plants worldwide (Agrios, 1988
In fungal phytopathogens, genes involved in pathogenicity have been identified by differential screening, by targeted deletion of candidate genes encoding a known physiological function, and by isolation of pathogenicity mutants. In Magnaporthe grisea, the causal agent of rice blast disease, darkly melanized appressoria similar to those of Colletotrichum species are formed. Analysis of signaling pathways and pathogenesis-related processes is being performed by many laboratories worldwide. Signaling pathways, cAMP, and mitogen-activated protein kinase (MAPK) pathways have been shown to play pivotal roles for fungal pathogenesis (Dean, 1997
In this study, we demonstrate that peroxisomal function is a prerequisite for fungal pathogenicity. We identify a novel insertional mutant of C. lagenarium generated by REMI, which is nonpathogenic to the host plant. Analysis of this mutant revealed that the loss of pathogenicity was caused by the disruption of a gene that exhibits significant homology with PEX6. Expression studies of green fluorescent protein (GFP)–containing PTS1 (for peroxisomal targeting signal1) showed that import of peroxisomal matrix proteins was impaired in pex6-deleted mutants of C. lagenarium. Also, the finding that the mutants grew poorly on fatty acid suggested a defect in fatty acid
Isolation of ClaPEX6 REMI insertional mutagenesis was performed to isolate nonpathogenic mutants of C. lagenarium wild-type strain 104-T. Plasmid pCB1004 containing the hygromycin resistance gene (hph) with no homology with C. lagenarium DNA was used in REMI experiments. Isolated REMI transformants were tested for pathogenicity to host cucumber leaves. One mutant, X86, was identified that induced no lesions on cucumber leaves. DNA gel blot analysis revealed that a single copy plasmid was integrated into the genome of X86 (data not shown). A 6.8-kb HindIII fragment including both pCB1004 and the flanking genomic region was recovered from X86 by plasmid rescue. DNA gel blot analysis of the rescued genome fragment identified a restriction fragment length polymorphism between the wild-type and X86 genomic DNA digested with EcoRI (Figure 1 , lanes 1 and 2). The wild-type 104-T contained a 7.0-kb EcoRI fragment, whereas X86 contained a 5.0-kb fragment. The rescued plasmid was introduced into the wild type to reproduce an insertional mutation of X86. Among 80 hygromycin-resistant transformants, six were nonpathogenic on cucumber (data not shown). DNA gel blot analysis demonstrated that the nonpathogenic transformants possessed the recombination event with the genomic region contained in the rescued plasmid. Recombination in the nonpathogenic transformants resulted in the disappearance of the 7.0-kb EcoRI fragment observed in the wild type (Figure 1, lanes 3 and 4), whereas transformants retaining pathogenicity maintained the 7.0-kb EcoRI fragment of the wild type (Figure 1, lanes 5 and 6). These results demonstrated that the pathogenicity defect in X86 was attributable to the insertional mutation to the rescued genomic region.
The rescued genomic region was used to isolate genome clones from the cosmid library of C. lagenarium. Cosmid inserts (ApaI, 9 kb; HindIII, 5 kb; and PstI, 2.7 kb) were subcloned into pBluescript KS-II. Sequence analysis of the rescued fragment and subclones revealed the existence of a large open reading frame spanning the whole region of the rescued fragment. The open reading frame consists of three exons (181, 3367, and 619 bp) and two introns (55 and 60 bp). The introns were confirmed by sequencing of cDNAs amplified by reverse transcription–polymerase chain reaction (PCR) (data not shown). The deduced amino acid sequence revealed high similarity with the sequences of members of Pex6 involved in peroxisome biogenesis (Figure 2) . We designated this gene ClaPEX6. The deduced amino acid sequence of ClaPEX6 has high homology with Pex6 proteins from P. chrysogenum (50% identity), Pichia pastoris (36%), Yarrowia lipolytica (36%), Hansenula polymorpha (34%), Saccharomyces cerevisiae (34%), and human (31%). Also, the putative Pex6 ortholog of Arabidopsis (accession number AAD25809) showed 27% identity with ClaPex6. ClaPex6 has two AAA cassettes, although the first AAA cassette is not highly conserved. Each AAA cassette consists of putative ATP binding motifs, as described by Walker et al. (1982) : a Walker A motif (THRNIGKA and GPPGTGKT) and a Walker B motif (KHVE and DELD) (Prosite accession number PS00017). ClaPex6 also contains the AAA-protein family signature (VFVIGATNRPDLLDPALLR) (accession number PS00674). ClaPex6 showed high similarity to other Pex6 proteins in the C-terminal region, including conserved motifs, although homology with P. chrysogenum Pex6 was found through the entire sequence of ClaPex6 (Figure 2). The sequence of the rescued fragment revealed that the plasmid insertion into X86 occurred in a position of the ClaPEX6 gene corresponding to the C-terminal region (at amino acid 1178) (Figure 2).
Disruption of ClaPEX6 Strain X86 was not pathogenic on cucumber, indicating that the ClaPEX6 gene is involved in fungal pathogenicity. However, the insertional event in X86 occurred in a region near the C terminus of ClaPex6. This implies the possibility that ClaPex6 would not lose its function completely in strain X86. Therefore, we generated a ClaPEX6 deletion mutant (clapex6  ) by one-step gene replacement. A gene replacement vector, pGDPEX6, containing the hph gene and both the 5' and 3' flanking regions of ClaPEX6, was constructed (Figure 3A)
. The region deleted by homologous recombination contained 2.8 kb of the coding region of ClaPEX6, including the highly conserved domains mentioned above. pGDPEX6 was introduced into the wild-type strain 104-T, and transformants were selected on hygromycin-containing medium.
To screen for clapex6 strains, we first investigated their pathogenicity to host cucumber leaves. Conidial suspensions from transformants were spotted on detached cucumber leaves and incubated for 7 days. Of 30 transformants tested, 21 did not induce lesions (Figure 4)
. These nonpathogenic transformants formed very few lesions even after 2 weeks of incubation (data not shown). DNA gel blot analysis was performed with three nonpathogenic transformants (DPE1, DPE8, and DPE9) and two pathogenic transformants (DPE4 and DPE7) (Figure 3B). The wild-type isolate 104-T and the transformants DPE4 and DPE7 maintained a 6.9-kb BamHI fragment (Figure 3B, lanes 1, 5, and 6). DPE4 and DPE7 contained one or two additional bands, indicating ectopic integration. In contrast, the nonpathogenic transformants DPE1, DPE8, and DPE9 did not contain the 6.9-kb BamHI wild-type fragment, but they did contain a common 5.5-kb BamHI fragment, consistent with the length expected from a gene replacement event (Figure 3B, lanes 2 to 4). These results demonstrated that the ClaPEX6 gene was disrupted in the nonpathogenic strains DPE1, DPE8, and DPE9. We concluded that the ClaPEX6 gene was essential for the pathogenicity of C. lagenarium. The clapex6 strains grew efficiently on nutrient-rich medium, although the growth rate was slightly lower than that of the wild type. Strain DPE1 grew at average 2.87 ± 0.03 cm (diameter) on potato dextrose agar (PDA) plates for 7 days, whereas the wild type grew at 3.32 ± 0.33 cm. Mycelial colonies formed by the mutants on PDA were darkly melanized similar to the wild type (data not shown). The amount of conidia produced by the clapex6 mutants was similar to that produced by the wild type (data not shown). These results indicate that the ClaPEX6 gene is dispensable for both growth and conidiation on nutrient-rich medium.
Appressoria Formed by clapex6 Mutants Are Defective in Host Penetration The clapex6 strains germinated effectively on a glass surface, and their germ tubes differentiated into swollen appressoria, indicating that the clapex6 mutants retain the ability to undergo the early steps for appressorium formation. However, appressoria formed by the clapex6 mutants were not identical to those of the wild type. Appressoria of the clapex6 mutants were smaller than those of the wild-type strain (Figures 5A and 5B
, Table 1). Furthermore, melanization of appressoria in the clapex6 mutants was severely reduced (Figures 5A and 5B), although their mycelium was as darkly melanized as that of the wild type (data not shown). We investigated whether appressoria formed by clapex6 mutants are able to penetrate the host plant. Conidia of the wild-type 104-T formed darkly melanized appressoria on the host plant. Three to 4 days after inoculation, the appressoria formed penetration hyphae into the epidermal cells (Figure 5C). The clapex6 mutants germinated and formed appressoria effectively on the plant surface, but their appressoria were slightly smaller and showed a decrease in melanization, which was similar to the phenotype on a glass surface (Figures 5B and 5D). These phenotypes also were observed in the original mutant X86 produced by REMI mutagenesis (data not shown).
Appressoria formed by the clapex6 mutants had the ability to penetrate an artificial substrate, cellulose membrane, suggesting that the mutants retain basic appressorium-mediated penetration ability (data not shown). However, appressoria of the clapex6 mutants did not form any penetration hyphae into the host plant, indicating that the mutants have a defect in appressorial penetration of plants (Figure 5D). To assess the ability of the clapex6 mutants to grow invasively inside the host plant cells independent of penetration, the mutants were inoculated through wounded sites of the plants. As a result, the clapex6 mutants formed lesions on the wounded leaves effectively (data not shown). This indicated that the clapex6 mutants retained the ability for invasive growth in the host plant. From these results, we concluded that the pathogenicity defect in clapex6 mutants was caused by the failure of penetration into the host plant, which resulted from formation of nonfunctional appressoria.
A Defect in the Transport of PTS1-Containing Proteins in clapex6 Mutants
The transformants expressing GFP-PTS1 or GFP exhibited identical phenotypes in growth, conidiation, appressorium formation, and pathogenicity to those of corresponding parental strains (data not shown). Localization of GFP fluorescence was investigated in conidia of each strain by fluorescence microscopy (Figure 7) . When GFP-PTS1 was expressed in the wild type, abundant punctate fluorescence was observed clearly, with no fluorescence being observed in the cytosol (Figure 7C). These fluorescent dots of GFP-PTS1 were observed at the peripheral region of conidia more frequently than at the central region, which appeared to be consistent with the results of electron microscopic analysis (Figures 6 and 7C). Conversely, wild-type strains expressing GFP showed diffuse fluorescence without a punctate pattern (Figure 7A). Occasionally, several spots were found in conidia showing diffuse fluorescence when GFP was highly expressed. These results suggest that GFP-PTS1 is recognized correctly and transported into the peroxisomes in the wild type of C. lagenarium. In contrast, when GFP-PTS1 was expressed in the clapex6 mutant, punctate fluorescence was not observed. The clapex6 mutant expressing GFP-PTS1 showed diffuse fluorescence, which was similar to the case of GFP expression in the mutant (Figures 7B and 7D). Similar results were obtained from observations of hyphal structure in each strain (data not shown). These results indicated that organelles visualized by GFP-PTS1 represent peroxisomes and that clapex6 mutants are defective in the import of peroxisome matrix proteins containing the PTS1 signal. We investigated the subcellular distribution of peroxisomes labeled by GFP-PTS1 during appressorium formation. First, conidia formed germ tubes, and germ tubes differentiated into swollen appressoria accompanied by the formation of vacuoles inside conidia. Growth and fusion of vacuoles resulted in the movement of cytoplasm from conidia to appressorial cells. At this stage, the majority of peroxisomes were localized in appressoria, not in conidia (Figure 7E). On the other hand, the clapex6 mutant showed diffuse fluorescence in both conidia and appressoria (Figure 7F).
Roles of Peroxisomes for Appressorium-Mediated Infection Peroxisome organelles contribute in various ways to cellular metabolism. The -oxidation pathway for degradation of fatty acids to acetyl-CoA is one of the main metabolic pathways in peroxisomes. To investigate peroxisomal metabolic functions in clapex6 mutants, we assessed the -oxidation pathway in the mutants by using a lipid-containing medium (Figure 8)
. It has been reported in several yeast species that most peroxisome-defective mutants are unable to grow on fatty acids (such as oleate) as sole carbon sources because of their defects in -oxidation of fatty acids (Spong and Subramani, 1993; Nuttley et al., 1994). We first investigated the growth ability of the wild type on fatty acid medium containing Tween 80, of which oleic acid is the major component. The wild type was able to grow on this fatty acid medium. The growth rate of the wild type on the fatty acid medium was similar to that on glucose medium as a sole carbon source (Figure 8). This result indicates that the wild-type strain of C. lagenarium is able to use fatty acids by the -oxidation pathway of fatty acids. In contrast to the wild type, the clapex6 mutants were completely defective in growth on medium containing Tween 80 (Figure 8B), whereas they showed growth similar to the wild type on glucose medium (Figure 8A). They did not form any mycelia on the Tween 80 medium even after extended incubation (20 days; data not shown). These results strongly suggested that clapex6 mutants have a defect in the fatty acid -oxidation pathway in peroxisomes.
Acetyl-CoA produced by -oxidation is transported to the mitochondrion, and it replenishes intermediates of the citric acid cycle in S. cerevisiae (van Roermund et al., 1995, 1999). Because clapex6 mutants were defective in the -oxidation pathway, we hypothesized that the pathogenicity defect in clapex6 mutants would be the result of the inability to supply citric acid cycle intermediates by -oxidation in peroxisomes. The glycolytic pathway, which metabolizes glucose, supplies intermediates of the citric acid cycle independent of -oxidation in peroxisomes. Therefore, we investigated appressorium formation of the clapex6 mutants in the presence of glucose. The wild-type strain formed normal appressoria in 1 mM glucose solution (data not shown). When conidia of the clapex6 mutant DPE1 were incubated in the glucose solution, they formed appressoria whose size and melanization increased compared with appressoria formed without glucose (Figures 9A and 9B , Table 1). On the other hand, scytalone, an intermediate product of the melanin biosynthesis pathway, restored appressorial melanization but not the size of appressoria in the clapex6 mutant (Figure 9C, Table 1).
The finding that glucose restored the phenotype of appressoria in the clapex6 mutant suggested that -oxidation in peroxisomes of the wild-type strain supports the citric acid cycle during appressorium formation and contributes to the appressorium maturation steps. However, even in the glucose solution, the pigment intensity of melanin in the mutant appressoria still was lower than that of the wild type, suggesting that restoration by glucose was incomplete (Figures 5 and 9). To assess whether the addition of glucose can restore the pathogenicity of the clapex6 mutants, conidia of the wild type and the clapex6 mutant DPE1 suspended in water, 1 mM scytalone, or 1 mM glucose solution were tested for the ability to infect cucumber leaves (Table 1). The wild-type strain induced lesions efficiently when conidia were suspended in water, scytalone solution, or glucose solution (Table 1 and data not shown). DPE1 formed lesions only in 1% of inoculated spots with water or scytalone solution. In contrast, inoculated with glucose, DPE1 formed lesions in  20% of inoculated spots, indicating that glucose metabolism partially compensated for the roles of peroxisomes in appressorium-mediated plant infection (Table 1).
We identified the PEX6 ortholog gene (ClaPEX6) of the fungal pathogen C. lagenarium by molecular analysis of the nonpathogenic REMI mutant. The human PEX6 gene has been identified as a gene responsible for peroxisome biogenesis disorder of complementation group C (group 4 in the United States) (Tsukamoto et al., 1995 ). The C. lagenarium clapex6 strains generated by gene replacement showed growth and conidiation similar to those of the wild type on nutrient-rich medium. This demonstrates that C. lagenarium PEX6 is dispensable for growth and conidiation in nutrient-rich conditions. On the other hand, Kiel et al. (2000) suggested the possibility that PEX6 is necessary for cell viability in the filamentous fungus P. chrysogenum. In the original mutant X86, the plasmid was inserted into a position corresponding to the C-terminal region of ClaPex6. Sequencing of the rescued plasmid revealed that the inserted vector sequence produced an in-frame stop codon at 77 to 79 bp downstream from the insertion point in the ClaPEX6 gene of X86 (data not shown). This suggests that strain X86 would produce a truncated ClaPex6 protein that contains conserved domains, including two AAA cassettes and an AAA-protein signature. The finding that the phenotype of X86 is similar to that of clapex6 deletion mutants suggests an essential role of the C-terminal region of the Pex6 protein, although this region has no characterized motifs.
Analysis by GFP-PTS1 expression indicated that the import of PTS1-containing peroxisome matrix proteins into peroxisomes was impaired in clapex6 mutants. It has been demonstrated in several organisms that peroxisomal membrane proteins do not use the sorting machinery of peroxisome matrix proteins to reach their target organelle but follow alternative pathways (Baerends et al., 2000
Studies in human and P. pastoris have shown that PEX6 is required for the stability of the PTS1 receptor, indicating a direct role of PEX6 for matrix protein import (Dodt and Gould, 1996
Peroxisomal Metabolism for Appressorium-Mediated Plant Infection
From studies in other organisms, including yeast, acetyl-CoA produced by fatty acid
It is known that acetyl-CoA from
When conidia of fungal pathogens attach on host plant surfaces, they must be subjected to poor nutrient conditions. Under this environment, they have to precede morphological developments such as appressorium formation for infection. It is plausible, therefore, that conidia of fungal pathogens contain storage compounds (e.g., lipid bodies and glycogen) that would be used for infection-related morphological developments (Hawker and Madelin, 1976
Fungal Strains, Media, and Fungal Transformation Colletotrichum lagenarium (syn C. orbiculare) strain 104-T (stock culture of the laboratory of Plant Pathology, Kyoto University) was used as the wild-type strain. All C. lagenarium cultures were maintained on 3.9% (w/v) potato dextrose agar (PDA) medium (Difco, Detroit, MI) at 24°C in the dark. Fatty acid medium contained 1.6% yeast nitrogen base without amino acids (Difco), 1% NH4NO3, 0.5% (v/v) Tween 80, and 1.5% agar. pH was adjusted to 6.0 with Na2HPO4. Glucose medium contained 1.6% yeast nitrogen base without amino acids (Difco), 1% NH4NO3, 2% glucose, and 1.5% agar. Protoplast preparation and transformation of C. lagenarium were performed basically according to the method described previously (Kubo et al., 1991 ). In restriction enzyme–mediated integration (REMI) mutagenesis, vector pCB1004 containing the hph gene was transformed into fungal protoplasts with restriction enzymes. Strain X86 was identified from screening of transformants generated with 50 units of XhoI. Transformants with the hph gene or the bar gene were selected on regeneration medium containing 100 µg/mL hygromycin B (Wako Pure Chemicals, Osaka, Japan) and 25 µg/mL bialaphos (Meiji Seika Kaisha, Ltd., Tokyo, Japan), respectively.
Genomic DNA Gel Blot Analysis
Isolation of ClaPEX6 by Plasmid Rescue and Sequence Analysis
Plasmid Constructs The amplified product was digested with HindIII and ClaI and introduced into pCB1531 to produce pBAT. The SCD1 promotor region was amplified with primers SD1PNS (5'-CAGGTTGCGGCCGCGTGTTTTGCGGCAGTCC-3') and SD1PXBA (5'-CGGGTCTAGACTGATAGGTGGGATATT-3'). SD1PNS and SD1PXBA contain a ter-minal NotI site and XbaI site, respectively. The amplified product was digested with NotI and XbaI and introduced into pBAT to produce pBATP. The entire GFP (EGFP) open reading frame fragment with the PTS1 sequence (SKL) was amplified from pCB16EGFP (Y. Takano, E. Oshiro, and T. Okuno, unpublished data) with primers EGFPX (5'-GCCCTCTAGACAGACACAATGGTGAGCAAGGGCGAG-3') and GFPPTS1B (5'-GGCGGATCCTTACAGCTTCGACTTGTACAGCTC-GTCCAT-3') and introduced into the XbaI-BamHI site of pBATP, which resulted in pBAGFPPTS1. The intact GFP fragment was amplified with primers EGFPX and GFPSTOP (5'-GGCGGATCCTTACTT-GTACAGCTCGTCCAT-3') and introduced into pBATP to produce pBAGFP. Each PCR amplification was performed with KOD-Plus DNA polymerase (Toyobo, Tsuruga, Japan) according to the manufacturer's instructions.
Pathogenicity Tests
Microscopy
Electron Microscopy
GenBank Accession Numbers
We thank Kazuyuki Mise for valuable suggestions and support during the course of this work. We acknowledge Ralph A. Dean and Thomas K. Mitchell for critical reading of the manuscript. We are grateful to Nobuyuki Fuchigami for providing bialaphos and Yasuyuki Kubo for providing scytalone. This work was supported in part by Grant 10760031 from the Ministry of Education, Science, Sports, and Culture of Japan and a Grant-in-Aid (JSPS-RFTF96L00603) from the Research for the Future program of the Japan Society for the Promotion of Science. Received March 2, 2001; accepted June 4, 2001.
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Abstract
INTRODUCTION
-32P-dCTP (Amersham Pharmacia Biotech, Buckinghamshire, UK) with the BcaBEST labeling kit (Takara, Ohtsu, Japan) or labeled with digoxigenin-dUTP (Boehringer Mannheim) with the BcaBEST digoxigenin labeling kit (Takara). DNA gel blot analysis was performed as described previously (Takano et al., 1997a
