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 responses. 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. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

Wheat stem rust recorded for the first time in decades in Ireland.

Wheat stem rust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), occurs in most wheat-growing areas worldwide, and, in western Europe since 2013, has started to re-emerge after many decades of absence. Following this trend across western Europe, in 2020, we also detected and recorded wheat stem rust for the first time in five decades in experimental plots across five locations in Ireland. To examine the potential origin of the Irish Pgt infection in 2020, we carried out transcriptome sequencing on 12 Pgt-infected wheat samples collected across Ireland and compared these to 76 global P. graminis isolates. This analysis identified a close genetic relationship between the Irish Pgt isolates and those from Ethiopia collected in 2015 after a severe stem rust epidemic caused by the TKTTF Pgt race, and with the UK-01 Pgt isolate that was previously assigned to the TKTTF race. Subsequent pathology-based race profiling designated two Irish isolates and recent UK and French Pgt isolates to the TKTTF Pgt race group. This suggests that the Irish Pgt occurrence most probably originated from recent long-distance windborne dispersal of Pgt urediniospores from neighbouring countries in Europe where we confirmed the Pgt TKTTF race continues to be prevalent. The identification of wheat stem rust in Ireland at multiple locations in 2020 illustrates that the disease can occur in Ireland and emphasizes the need to re-initiate local monitoring for this re-emergent threat to wheat production across western Europe.

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

Plant pathogens secrete 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 putative 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.

A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity.

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Telomeres and a repeat-rich chromosome encode effector gene clusters in plant pathogenic Colletotrichum fungi.

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in many commercially important plants. Closely related strains have different levels of pathogenicity on hosts despite their close phylogenetic relationship. To gain insight into the genetics underlying these differences, we generated and annotated whole-genome assemblies of multiple isolates of C. fructicola (Cf) and C. siamense (Cs), as well as three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum with different pathogenicity on strawberry. Based on comparative genomics, we identified accessory regions with a high degree of conservation in strawberry-pathogenic Cf, Cs and Ca strains. These regions encode homologs of pathogenicity-related genes known as effectors, organized in syntenic gene clusters, with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca revealed the association of related accessory effector gene clusters with telomeres and repeat-rich chromosomes and provided evidence of exchange between these two genomic compartments. In addition, expression analysis indicated that orthologues in syntenic gene clusters showed a tendency for correlated gene expression during infection. These data provide insight into mechanisms by which Colletotrichum genomes evolve, acquire and organize effectors.

Genomic Plasticity Mediated by Transposable Elements in the Plant Pathogenic Fungus Colletotrichum higginsianum.

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts.

Method for Assessing Virulence of Colletotrichum higginsianum on Arabidopsis thaliana Leaves Using Automated Lesion Area Detection and Measurement.

The plant pathogenic fungus, Colletotrichum higginsianum is widely used to understand infection mechanisms, as it infects the model plant Arabidopsis thaliana. To determine the virulence of C. higginsianum, several methods have been developed, such as disease reaction scoring, lesion measurement, entry rate assays, and relative fungal biomass assays using real-time quantitative PCR. Although many studies have taken advantage of these methods, they have shortcomings in terms of objectivity, time, or cost. Here, we show a lesion area detection method applying ImageJ color thresholds to images of A. thaliana leaves infected by C. higginsianum. This method can automatically detect multiple lesions in a short time without the requirement for special equipment and measures lesion areas in a standardized way. This high throughput technique will aid better understanding of plant immunity and pathogenicity and contribute to reproducibility of assays.

Genus-Wide Comparative Genome Analyses of Colletotrichum Species Reveal Specific Gene Family Losses and Gains during Adaptation to Specific Infection Lifestyles.

Members from Colletotrichum genus adopt a diverse range of lifestyles during infection of plants and represent a group of agriculturally devastating pathogens. In this study, we present the draft genome of Colletotrichum incanum from the spaethianum clade of Colletotrichum and the comparative analyses with five other Colletotrichum species from distinct lineages. We show that the C. incanum strain, originally isolated from Japanese daikon radish, is able to infect both eudicot plants, such as certain ecotypes of the eudicot Arabidopsis, and monocot plants, such as lily. Being closely related to Colletotrichum species both in the graminicola clade, whose members are restricted strictly to monocot hosts, and to the destructivum clade, whose members are mostly associated with dicot infections, C. incanum provides an interesting model system for comparative genomics to study how fungal pathogens adapt to monocot and dicot hosts. Genus-wide comparative genome analyses reveal that Colletotrichum species have tailored profiles of their carbohydrate-degrading enzymes according to their infection lifestyles. In addition, we show evidence that positive selection acting on secreted and nuclear localized proteins that are highly conserved may be important in adaptation to specific hosts or ecological niches.