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Makoto Asakura Kae Yoshino Alison M. Hill Yasuyuki Kubo Yasuyoshi Sakai Yoshitaka Takano 《Fungal genetics and biology : FG & B》2012,49(11):967-975
The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures. 相似文献
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《Experimental mycology》1983,7(3):208-215
Melanin biosynthesis of appressoria inColletotrichum lagenaru was studied using color mutants. Mutant 8015 ofC. lagenarium obtained byN-methyl-N′-nitro-N-nitroso-guanidine treatment formed a red-brown colony and secreted a substance which restored black coloration to colonies of albino mutants. This substance was identified as scytalone (3,4-dihydro-3,6,8-trihydroxy-1(2H)naphthalenone). Nineteen albino mutants formed colorless appressoria, but in the presence of 0.75 mM scytalone, the albino mutants formed darkly pigmented appressoria indistinguishable from those of the parent strain. Furthermore, the time course of appressorial pigmentation of albino mutants in the presence of scytalone was the same as that of the parent strain. On nitrocellulose membranes and host cucumber cotyledons, the colorless appressoria of albino mutants germinated laterally to form secondary appressoria, and consequently had little ability to form penetration hyphae. In the presence of 0.75 mM scytalone, however, the pigmented appressoria penetrated nitrocellulose membranes and host cucumber cell walls similarly to those of the parent strain. From these results, we conclude that scytalone is a normal precusor of melanin in appressoria, and that appressorial pigmentation is essential for the formation of penetration hyphae inC. lagenarium. 相似文献
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Multiple Contributions of Peroxisomal Metabolic Function to Fungal Pathogenicity in Colletotrichum lagenarium
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In Colletotrichum lagenarium, which is the causal agent of cucumber anthracnose, PEX6 is required for peroxisome biogenesis and appressorium-mediated infection. To verify the roles of peroxisome-associated metabolism in fungal pathogenicity, we isolated and functionally characterized ICL1 of C. lagenarium, which encodes isocitrate lyase involved in the glyoxylate cycle in peroxisomes. The icl1 mutants failed to utilize fatty acids and acetate for growth. Although Icl1 has no typical peroxisomal targeting signals, expression analysis of the GFP-Icl1 fusion protein indicated that Icl1 localizes in peroxisomes. These results indicate that the glyoxylate cycle that occurs inside the peroxisome is required for fatty acid and acetate metabolism for growth. Importantly, in contrast with the pex6 mutants that form nonmelanized appressoria, the icl1 mutants formed appressoria that were highly pigmented with melanin, suggesting that the glyoxylate cycle is not essential for melanin biosynthesis in appressoria. However, the icl1 mutants exhibited a severe reduction in virulence. Appressoria of the icl1 mutants failed to develop penetration hyphae in the host plant, suggesting that ICL1 is involved in host invasion. The addition of glucose partially restored virulence of the icl1 mutant. Heat shock treatment of the host plant also enabled the icl1 mutants to develop lesions, implying that the infection defect of the icl1 mutant is associated with plant defense. Together with the requirement of PEX6 for appressorial melanization, our findings suggest that peroxisomal metabolic pathways play functional roles in appressorial melanization and subsequent host invasion steps, and the latter step requires the glyoxylate cycle. 相似文献
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Kelch repeat protein Clakel2p and calcium signaling control appressorium development in Colletotrichum lagenarium 总被引:1,自引:0,他引:1
Kelch repeat proteins are important mediators of fundamental cellular functions and are found in diverse organisms. However, the roles of these proteins in filamentous fungi have not been characterized. We isolated a kelch repeat-encoding gene of Colletotrichum lagenarium ClaKEL2, a Schizosaccharomyces pombe tea1 homologue. Analysis of the clakel2 mutant indicated that ClaKEL2 was required for the establishment of cellular polarity essential for proper morphogenesis of appressoria and that there is a plant signal-specific bypass pathway for appressorium development which circumvents ClaKEL2 function. Clakel2p was localized in the polarized region of growing hyphae and germ tubes, and the localization was disturbed by a microtubule assembly blocker. The clakel2 mutants formed abnormal appressoria, and those appressoria were defective in penetration hypha development into cellulose membranes, an artificial model substrate for fungal infection. Surprisingly, the clakel2 mutants formed normal appressoria on the host plant and retained penetration ability. Normal appressorium formation on the artificial substrate by the clakel2 mutants was restored when cells were incubated in the presence of CaCl2 or exudates from cucumber cotyledon. Furthermore, calcium channel modulators inhibited restoration of normal appressorium formation. These results suggest that there could be a bypass pathway that transduces a plant-derived signal for appressorium development independent of ClaKEL2 and that a calcium signal is involved in this transduction pathway. 相似文献
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Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare 总被引:1,自引:0,他引:1
Makoto Asakura Sachiko Ninomiya Miki Sugimoto Masahide Oku Shun-ichi Yamashita Tetsuro Okuno Yasuyoshi Sakai Yoshitaka Takano 《The Plant cell》2009,21(4):1291-1304
The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare. 相似文献
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Takano Y Komeda K Kojima K Okuno T 《Molecular plant-microbe interactions : MPMI》2001,14(10):1149-1157
Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium. 相似文献
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In Colletotrichum lagenarium, which is the causal agent of cucumber anthracnose, PEX6 is required for peroxisome biogenesis and appressorium-mediated infection. To verify the roles of peroxisome-associated metabolism in fungal pathogenicity, we isolated and functionally characterized ICL1 of C. lagenarium, which encodes isocitrate lyase involved in the glyoxylate cycle in peroxisomes. The icl1 mutants failed to utilize fatty acids and acetate for growth. Although Icl1 has no typical peroxisomal targeting signals, expression analysis of the GFP-Icl1 fusion protein indicated that Icl1 localizes in peroxisomes. These results indicate that the glyoxylate cycle that occurs inside the peroxisome is required for fatty acid and acetate metabolism for growth. Importantly, in contrast with the pex6 mutants that form nonmelanized appressoria, the icl1 mutants formed appressoria that were highly pigmented with melanin, suggesting that the glyoxylate cycle is not essential for melanin biosynthesis in appressoria. However, the icl1 mutants exhibited a severe reduction in virulence. Appressoria of the icl1 mutants failed to develop penetration hyphae in the host plant, suggesting that ICL1 is involved in host invasion. The addition of glucose partially restored virulence of the icl1 mutant. Heat shock treatment of the host plant also enabled the icl1 mutants to develop lesions, implying that the infection defect of the icl1 mutant is associated with plant defense. Together with the requirement of PEX6 for appressorial melanization, our findings suggest that peroxisomal metabolic pathways play functional roles in appressorial melanization and subsequent host invasion steps, and the latter step requires the glyoxylate cycle. 相似文献
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Atg26-mediated pexophagy and fungal phytopathogenicity 总被引:1,自引:0,他引:1
《Autophagy》2013,9(7):1041-1042
Colletotrichum orbiculare is a plant pathogenic fungus that causes disease on cucumber plants. A homologue of ATG26 (CoATG26) was identified as the gene involved in pathogenesis. The peroxisomes are degraded via pexophagy during formation of an infection structure called the appressorium of C. orbiculare. The Coatg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step. Importantly, the autophagic degradation of peroxisomes was significantly delayed in the appressoria of the Coatg26 mutant. Domain and localization analysis of CoAtg26 also demonstrated a strong correlation of functional pexophagy with pathogenicity. Furthermore, in contrast to the Coatg26 mutant, the Coatg8 mutant, defective in the entire autophagic pathway, could not form normal appressoria in the earlier steps of morphogenesis. These results indicate that CoAtg26-mediated pexophagy plays critical roles in host plant invasion. 相似文献
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Hiromasa Saitoh Shizuko Fujisawa Chikako Mitsuoka Akiko Ito Akiko Hirabuchi Kyoko Ikeda Hiroki Irieda Kae Yoshino Kentaro Yoshida Hideo Matsumura Yukio Tosa Joe Win Sophien Kamoun Yoshitaka Takano Ryohei Terauchi 《PLoS pathogens》2012,8(5)
To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively. 相似文献
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Shigeyuki Tanaka Nobuaki Ishihama Hirofumi Yoshioka Aurélie Huser Richard O'Connell Gento Tsuji Seiji Tsuge Yasuyuki Kubo 《The Plant cell》2009,21(8):2517-2526
Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance. 相似文献
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Kei Hiruma Mariko Onozawa-Komori Fumika Takahashi Makoto Asakura Pawe? Bednarek Tetsuro Okuno Paul Schulze-Lefert Yoshitaka Takano 《The Plant cell》2010,22(7):2429-2443
When faced with nonadapted fungal pathogens, Arabidopsis thaliana mounts nonhost resistance responses, which typically result in the termination of early pathogenesis steps. We report that nonadapted anthracnose fungi engage two alternative entry modes during pathogenesis on leaves: turgor-mediated invasion beneath melanized appressoria, and a previously undiscovered hyphal tip–based entry (HTE) that is independent of appressorium formation. The frequency of HTE is positively regulated by carbohydrate nutrients and appears to be subject to constitutive inhibition by the fungal mitogen-activated protein kinase (MAPK) cascade of MAPK ESSENTIAL FOR APPRESSORIUM FORMATION1. The same MAPK cascade is essential for appressorium formation. Unexpectedly, the Arabidopsis indole glucosinolate pathway restricts entry of the nonadapted anthracnose fungi only when these pathogens employ HTE. Arabidopsis mutants defective in indole glucosinolate biosynthesis or metabolism support the initiation of postinvasion growth of nonadapted Colletotrichum gloeosporioides and Colletotrichum orbiculare. However, genetic disruption of Colletotrichum appressorium formation does not permit HTE on host plants. Thus, Colletotrichum appressoria play a critical role in the suppression of preinvasion plant defenses, in addition to their previously described role in turgor-mediated plant cell invasion. We also show that HTE is the predominant morphogenetic response of Colletotrichum at wound sites. This implies the existence of a fungal sensing system to trigger appropriate morphogenetic responses during pathogenesis at wound sites and on intact leaf tissue. 相似文献
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The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach 相似文献
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Yoshihiro Takikawa Koji Kakutani Teruo Nonomura Yoshinori Matsuda Hideyoshi Toyoda 《Mycoscience》2011,52(3):204-209
In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the
germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA
internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied
on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than
90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all
substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies
without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however,
all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous
appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium.
Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these
two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape. 相似文献
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