Entry mode-dependent function of an indole glucosinolate pathway in Arabidopsis for nonhost resistance against anthracnose pathogens.

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.

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare.

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 ß-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 ß-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 ß-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.

Atg26-mediated pexophagy and fungal phytopathogenicity.

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.

SPM1 encoding a vacuole-localized protease is required for infection-related autophagy of the rice blast fungus Magnaporthe oryzae.

SPM1, encoding a putative subtilisin-like protease, is involved in pathogenicity of the rice blast fungus Magnaporthe oryzae, but its detailed function remains unknown. Here, we report that SPM1 encodes a vacuole-localized protease that is a critical component for autophagy during the infection process of M. oryzae. Detailed phenotypic analysis of targeted disruption mutants of SPM1 revealed that the mutants have pleiotropic defects in infection-related steps including germination, appressorium formation, host invasion and postinvasive growth, indicating the requirement of Spm1 function for the broad phase of infection. It has been shown that the Spm1 homolog of yeast functions in autophagy, the degradation machinery mediated by vacuoles, implying the involvement of Spm1 in autophagy in M. oryzae. In-gel protease activity assay of the recombinant Spm1 protein indicated that Spm1 had a protease activity. An Spm1-GFP fusion protein was detected inside vacuoles of fungal cells, indicating that Spm1 is a protease localized in vacuoles. Furthermore, degradation of putative autophagic bodies was retarded in vacuoles of the spm1 mutant. These data strongly suggest that SPM1-encoded protease functions in autophagy required for the pathogenicity of M. oryzae.

Atg26-mediated pexophagy is required for host invasion by the plant pathogenic fungus Colletotrichum orbiculare.

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.

Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium.

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.