Yoshitaka Takano

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae.

To subvert rice (Oryza sativa) host defenses, the devastating ascomycete fungus pathogen Magnaporthe oryzae produces a battery of effector molecules, including some with avirulence (AVR) activity, which are recognized by host resistance (R) proteins resulting in rapid and effective activation of innate immunity. To isolate novel avirulence genes from M. oryzae, we examined DNA polymorphisms of secreted protein genes predicted from the genome sequence of isolate 70-15 and looked for an association with AVR activity. This large-scale study found significantly more presence/absence polymorphisms than nucleotide polymorphisms among 1032 putative secreted protein genes. Nucleotide diversity of M. oryzae among 46 isolates of a worldwide collection was extremely low (θ = 8.2 × 10−5), suggestive of recent pathogen dispersal. However, no association between DNA polymorphism and AVR was identified. Therefore, we used genome resequencing of Ina168, an M. oryzae isolate that contains nine AVR genes. Remarkably, a total of 1.68 Mb regions, comprising 316 candidate effector genes, were present in Ina168 but absent in the assembled sequence of isolate 70-15. Association analyses of these 316 genes revealed three novel AVR genes, AVR-Pia, AVR-Pii, and AVR-Pik/km/kp, corresponding to five previously known AVR genes, whose products are recognized inside rice cells possessing the cognate R genes. AVR-Pia and AVR-Pii have evolved by gene gain/loss processes, whereas AVR-Pik/km/kp has evolved by nucleotide substitutions and gene gain/loss.

The Colletotrichum lagenarium MAP Kinase Gene CMK1 Regulates Diverse Aspects of Fungal Pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

STRUCTURAL ANALYSIS OF PKS1, A POLYKETIDE SYNTHASE GENE INVOLVED IN MELANIN BIOSYNTHESIS IN COLLETOTRICHUM LAGENARIUM

Albino mutants (Pks−) of Colletotrichum lagenarium form nonmelanized appressoria and possess little penetrating ability on the host plant. The defect in albino mutant 79215 (Pks−) is considered to lie in pentaketide biosynthesis and/or pentaketide cyclization during melanin biosynthesis. The cosmid pAC7, carrying the PKS1 gene, when transformed into the albino mutant restores the wild-type melanin phenotype. We have determine the DNA sequence and the transcriptional organization of the PKS1 gene. The PKS1 gene contains one open reading frame, consisting of 3 exons separated by two short introns. The predicted PKS1 polypeptide consists of 2187 amino acids and shows significant similarities with other polyketide synthases, particularly that encoded by wA in Aspergillus nidulans, involved in conidial pigmentation. The PKS1 gene contains highly conserved β-ketoacyl synthase, acetyl/malonyl transferase, and acyl carrier protein domains. We propose that the C. lagenarium PKS1 gene encodes a polyketide synthase involved in melanin biosynthesis.

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild‐type fungus was not able to infect the host plant because of a failure of appressorium‐mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1‐GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1‐type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.

Peroxisomal Metabolic Function Is Required for Appressorium-Mediated Plant Infection by Colletotrichum lagenarium

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 β-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.

Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi

Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray. Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes. Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.

Cloning and characterization of a melanin biosynthetic THR1 reductase gene essential for appressorial penetration of Colletotrichum lagenarium

Melanin biosynthesis of Colletotrichum lagenarium is essential for appressorial penetration of the host plant. A melanin deficient mutant 9141 (Thr-) has a defect in the conversion of 1,3,8-trihydroxynaphthalene to vermelone in the melanin biosynthetic pathway. The mutant formed nonmelanized appressoria and had little infectivity on cucumber leaves. A cosmid clone pCR1 was selected from a cosmid library of wild-type C. lagenarium by means of a heterologous probe BRM2, one of the clustered genes involved in melanin biosynthesis of Alternaria alternata. pCR1 transformed the Thr- mutant 9141 to wild-type phenotype. A DNA fragment (THR1) homologous to BRM2 was subcloned from pCR1 and the nucleotide sequence determined. THR1 contains one open reading frame that encodes a protein of 282 amino acids. A transformant resulting from gene disruption showed a light brown phenotype different from the dark brown phenotype of the wild-type 104-T. The transformant formed nonmelanized appressoria and had little infectivity. The THR1 amino acid sequence contains a region highly similar to the Ver1 gene involved in the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis by Aspergillus parasiticus and to the T4HN reductase gene involved in the conversion of 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone in melanin biosynthesis by Magnaporthe grisea. Expression of the THR1 gene during spore germination of C. lagenarium was detected by RNA blotting. We propose that the C. lagenarium THR1 gene encodes a reductase involved in conversion of 1,3,8-trihydroxynaphthalene to vermelone.

Nonhost resistance in Arabidopsis-colletotrichum interactions acts at the cell periphery and requires actin filament function

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

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.