Long non-coding RNAs (lncRNAs) play an important role in biological processes but regulation and function of lncRNAs remain largely unelucidated, especially in fungi. Ustilaginoidea virens is an economically important fungus causing a devastating disease of rice. By combining microscopic and RNAseq analyses, we comprehensively characterized lncRNAs of this fungus in infection and developmental processes and defined four serial typical stages. RNA-seq analyses revealed 1724 lncRNAs in U. virens, including 1084 long intergenic non-coding RNAs (lincRNAs), 51 intronic RNAs (incRNAs), 566 natural antisense transcripts (lncNATs) and 23 sense transcripts. Gene Ontology enrichment of differentially expressed lincRNAs and lncNATs demonstrated that these were mainly involved in transport-related regulation. Functional studies of transport-related lncRNAs revealed that UvlncNAT-MFS, a cytoplasm localized lncNAT of a putative MFS transporter gene, UvMFS, could form an RNA duplex with UvMFS and was required for regulation of growth, conidiation and various stress responses.Our results were the first to elucidate the lncRNA profiles during infection and development of this important phytopathogen U. virens. The functional discovery of the novel lncRNA, UvlncNAT-MFS, revealed the potential of lncRNAs in regulation of life processes in fungi.

Colletotrichum higginsianum is a hemibiotrophic ascomycetous fungus that causes economically important anthracnose diseases on numerous monocot and dicot crops worldwide. As a model pathosystem, the Colletotrichum–Arabidopsis interaction has the significant advantage that both organisms can be manipulated genetically. The goal of this review is to provide an overview of the system and to point out recent significant studies that update our understanding of the pathogenesis of C. higginsianum and resistance mechanisms of Arabidopsis against this hemibiotrophic fungus. The genome sequence of C. higginsianum has provided insights into how genome structure and pathogen genetic variability has been shaped by transposable elements, and allows systematic approaches to longstanding areas of investigation, including infection structure differentiation and fungal–plant interactions. The Arabidopsis-Colletotrichum pathosystem provides an integrated system, with extensive information on the host plant and availability of genomes for both partners, to illustrate many of the important concepts governing fungal–plant interactions, and to serve as an excellent starting point for broad perspectives into issues in plant pathology.

Colletotrichum higginsianum is a widely distributed fungus attacking many cruciferous species. To investigate pathogenic mechanisms of the pathogen on the host Arabidopsis thaliana, we screened and obtained a virulence-deficient mutant Ch-1-T513 in a T-DNA insertion mutant library of C. higginsianum. The mutant Ch-1-T513 produced yellow colony centers with distorted multi-branching hyphal tips as well as producing few conidia. Heavily swollen hyphae in the mutant could be observed, and intra-hyphal hyphae were found to be formed in the balloon-shaped hyphae. The mutant failed to produce lesions on 12-day-old Arabidopsis seedlings, and invasive hyphae did not differentiate into large primary and thin secondary hyphae after appressorial formation on Arabidopsis leaves, but formed abundant bulbous hyphae in epidermal cells. Southern blot analysis showed Ch-1-T513 had double-site T-DNA integrations. The mutant had insertions upstream of genes for a major facilitator superfamily (MFS) transporter, ChMfs1 and an aldo/keto reductase, ChAkr. Complementation experiments by transforming genomic sequences from a wild-type strain into the insertion mutant demonstrated that ChMfs1 is involved in the Ch-1-T513 phenotype. The complementation strain C-ChMfs1-1 exhibited normal hyphal morphology, conidiation, and pathogenicity identical to the wild-type. The results demonstrate that ChMfs1 is involved in intra-hyphal hyphae production, conidiation, and pathogenicity in C. higginsianum. To our knowledge, this is the first report of a MFS transporter gene in a phytopathogenic fungus associated with intra-hyphal hyphae formation, playing a key role in infection of its plant host.

Colletotrichum higginsianum is an important fungal pathogen causing anthracnose disease of cruciferous plants. In this study, we characterized a putative orthologue of yeast SPE1 in C. higginsianum, named ChODC. Deletion mutants of ChODC were defective in hyphal and conidial development. Importantly, deletion of ChODC significantly affected appressoriummediated penetration in C. higginsianum. However, polyamines partially restore appressorium function and virulence indicating that loss of ChODC caused significantly decreased virulence by the crosstalk between polyamines and other metabolic pathways. Subsequently, transcriptomic and metabolomic analyses demonstrated that ChODC played an important role in metabolism of various carbon and nitrogen compounds including amino acids, carbohydrates and lipids. Along with these clues, we found deletion of ChODC affected glycogen and lipid metabolism, which were important for conidial storage utilization and functional appressorium formation. Loss of ChODC affected the mTOR signalling pathway via modulation of autophagy. Interestingly, cAMP treatment restored functional appressoria to the ΔChODC mutant, and rapamycin treatment also stimulated formation of functional appressoria in the ΔChODC mutant. Overall, ChODC was associated with the polyamine biosynthesis pathway, as a mediator of cAMP and mTOR signalling pathways to regulate appressorium function. Our study provides evidence of a link between ChODC and the cAMP signalling pathway and defines a novel mechanism by which ChODC regulates infection-associated autophagy and plant infection by fungi.

Colletotrichum higginsianum is an important hemibiotrophic phytopathogen that causes crucifer anthracnose in various regions of the world. In many plant-pathogenic fungi, the Ste11-Ste7-Fus3/Kss1 kinase pathway is essential to pathogenicity and various plant infection processes. To date, the role of ChSte7 in C. higginsianum encoding a MEK orthologue of Ste7 in Saccharomyces cerevisiae has not been elucidated. In this report, we investigated the function of ChSte7 in the pathogen. The ChSte7 is predicted to encode a 522-amino-acid protein with a S_TKc conserved domain that shares 44% identity with Ste7 in S. cerevisiae. ChSte7 disruption mutants showed white colonies with irregularly shaped edges and extremely decreased growth rates and biomass productions. The ChSte7 disruption mutants did not form appressoria and showed defects in pathogenicity on leaves of Arabidopsis thaliana. When inoculated onto wounded leaf tissues, the ChSte7 disruption mutants grew only on the surface of host tissues but failed to cause lesions beyond the wound site. In contrast, both the wild-type and complementation strains showed normal morphology, produced appressoria, and caused necrosis on leaves of Arabidopsis. Analysis with qRT-PCR suggested that ChSte7 was highly expressed during the late stages of infection. Taken together, our results demonstrate that ChSte7 is involved in regulation of vegetative growth, appressorial formation of C. higginsianum, and postinvasive growth in host tissues.

Prohibitins are highly conserved eukaryotic proteins in mitochondria that function in various cellular processes. The roles of prohibitins in fungal virulence and their regulatory mechanisms are still unknown. Here, we identified the prohibitins ChPhb1 and ChPhb2 in a plant pathogenic fungus Colletotrichum higginsianum and investigated their roles in the virulence of this anthracnose fungus attacking crucifers. We demonstrate that ChPhb1 and ChPhb2 are required for the proper functioning of mitochondria, mitophagy and virulence. ChPhb1 and ChPhb2 interact with the autophagy-related protein ChATG24 in mitochondria, and ChATG24 shares similar functions with these proteins in mitophagy and virulence, suggesting that ChATG24 is involved in prohibitin-dependent mitophagy. ChPhb1 and ChPhb2 modulate the translocation of ChATG24 into mitochondria during mitophagy. The role of ChATG24 in mitophagy is further confirmed to be conserved in plant pathogenic fungi. Our study presents that prohibitins regulate fungal virulence by mediating ATG24-assisted mitophagy.

The fungal pathogen, Colletotrichum higginsianum, causes a disease called anthracnose on various cruciferous plants. Here, we characterized a Saccharomyces cerevisiae CDC25 ortholog in C. higginsianum, named ChCDC25 (CH063_04363). The ChCDC25 deletion mutants were defective in mycelial growth, conidiation, conidial germination, appressorial formation, and invasive hyphal growth on Arabidopsis leaves, resulting in loss of virulence. Furthermore, deletion of ChCDC25 led to increased sensitivity to cell wall stress and resulted in resistance to osmotic stress. Exogenous cyclic adenosine monophosphate (cAMP) and IBMX treatments were able to induce appressorial formation in the ChCDC25 mutants, but abnormal germ tubes were still formed. The results implied that ChCDC25 is involved in pathogenicity by regulation of cAMP signaling pathways in C. higginsianum. More importantly, we found that ChCDC25 may interact with Ras2 and affects Ras2 protein abundance in C. higginsianum. Taken together, ChCDC25 regulates infection-related morphogenesis and pathogenicity of C. higginsianum. This is the first report to reveal functions of a CDC25 ortholog in a hemibiotrophic phytopathogen.

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