Biology subjects

Kumakura, N.

Publications and source records attributed to Kumakura, N..

6 recordsLinked to original sources

bioRxiv2024

Dihydroxyhexanoic acid biosynthesis controls turgor in pathogenic fungi

Many plant pathogenic fungi penetrate host surfaces mechanically, using turgor pressure generated by appressoria, specialized infection cells. These appressoria develop semipermeable cell walls and accumulate osmolytes internally to create turgor by osmosis. While melanin is known to be important for turgor generation, the mechanism for wall semipermeability has remained unclear. Here we identify PKS2 and PBG13, by reverse genetics, as crucial for forming the semipermeable barrier in anthracnose and rice blast fungi. These genes encode enzymes that synthesize 3,5-dihydroxyhexanoic acid polymers essential for the cell wall properties. Deleting these enzymes impairs cell wall porosity, abolishing turgor and pathogenicity without affecting melanization. Our findings uncover a novel mechanism of turgor generation, linking enzyme function to pathogen penetration and disease potential, presenting new targets for disease control.

plant biology↗

bioRxiv2023

Efficient multiple gene knock-out in Colletotrichum higginsianum via CRISPR-Cas9 ribonucleoprotein and URA3- based marker recycling

Colletotrichum higginsianum is a hemibiotrophic pathogen that causes anthracnose disease on crucifer hosts, including Arabidopsis thaliana. Despite the availability of genomic and transcriptomic information and the ability to transform both organisms, identifying C. higginsianum genes involved in virulence has been challenging due to their recalcitrance to gene targeting and redundancy of virulence factors. To overcome these obstacles, we developed an efficient method for multiple gene disruption in C. higginsianum by combining CRISPR-Cas9 and URA3-based marker recycling systems. Our method significantly increased the efficiency of gene knock-out via homologous recombination by introducing genomic DNA double-strand breaks. We demonstrated the applicability of the URA3-based marker recycling system for multiple gene targeting in the same strain. Using our technology, we successfully targeted two melanin biosynthetic genes, SCD1 and PKS1, which resulted in deficiency in melanisation and pathogenicity in the mutants. Our findings demonstrate the effectiveness of our developments in analysing virulence factors in C. higginsianum, thus accelerating research on plant-fungus interactions.

microbiology↗

bioRxiv2022

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier of antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocalgates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to Ophiocordyceps genus and its eIF4A, a molecular target of rocaglates, contains a amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered Colletotrichum orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

molecular biology↗

bioRxiv2021

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

microbiology↗

bioRxiv2021

The Conserved Colletotrichum spp. Effector CEC3 Induces Nuclear Expansion and Cell Death in Plants

Plant pathogens secrete small proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

microbiology↗

bioRxiv2020

Subtelomeric regions and a repeat-rich chromosome harbor multicopy effector gene clusters with variable conservation in multiple plant pathogenic Colletotrichum species

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in a wide range of commercially important plants. To provide an in-depth overview of its diversity, we sequenced the genomes of fungi belonging to this group, including multiple strains of C. fructicola (Cf) and C. siamense (Cs), as well as representatives of three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum. Comparisons between multiple Cf and Cs strains led to the identification of accessory regions that show variable conservation in both lineages. These accessory regions encode effector candidate genes, including homologs of previously characterized effectors, organized in clusters of conserved synteny with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca strains revealed the association of such accessory effector gene clusters with subtelomeric regions and repeat-rich minichromosomes and provided evidence of gene transfer between these two genomic compartments. In addition, expression analysis indicated that paralogs associated with clusters of conserved synteny showed a tendency for correlated gene expression. These data highlight the importance of subtelomeric regions and repeat-rich chromosomes to the genome plasticity of Colletotrichum fungi.

genomics↗