Biology subjects

Dallery, J.-F.

Publications and source records attributed to Dallery, J.-F..

5 recordsLinked to original sources

bioRxiv2023

Complete genome of the Medicago anthracnose fungus, Colletotrichum destructivum, reveals a mini-chromosome-like region within a core chromosome.

Colletotrichum destructivum (Cd) is a phytopathogenic fungus causing significant economic losses on forage legume crops (Medicago and Trifolium species) worldwide. To gain insights into the genetic basis of fungal virulence and host specificity, we sequenced the genome of an isolate from M. sativa using long-read (PacBio) technology. The resulting genome assembly has a total length of 51.7 Mb and comprises 10 core chromosomes and two accessory chromosomes, all of which were sequenced from telomere to telomere. A total of 15,631 gene models were predicted, including genes encoding potentially pathogenicity-related proteins such as candidate secreted effectors (484), secondary metabolism key enzymes (110) and carbohydrate-active enzymes (619). Synteny analysis revealed extensive structural rearrangements in the genome of Cd relative to the closely-related Brassicaceae pathogen, C. higginsianum. In addition, a 1.2 Mb species-specific region was detected within the largest core chromosome of Cd that has all the characteristics of fungal accessory chromosomes (transposon-rich, gene-poor, distinct codon usage), providing evidence for exchange between these two genomic compartments. This region was also unique in having undergone extensive intra-chromosomal segmental duplications. Our findings provide insights into the evolution of accessory regions and possible mechanisms for generating genetic diversity in this asexual fungal pathogen. Impact statementColletotrichum is a large genus of fungal phytopathogens that cause major economic losses on a wide range of crop plants throughout the world. These pathogens vary widely in their host specificity and may have either broad or narrow host ranges. Here, we report the first complete genome of the alfalfa (Medicago sativa) pathogen, Colletotrichum destructivum, which will facilitate the genomic analysis of host adaptation and comparison with other members of the Destructivum species complex. We identified a species-specific 1.2 Mb region within chromosome 1 displaying all the hallmarks of fungal accessory chromosomes, which may have arisen through the integration of a mini-chromosome into a core chromosome and could be linked to the pathogenicity of this fungus. We show this region is also a focus for segmental duplications, which may contribute to generating genetic diversity for adaptive evolution. Finally, we report infection by this fungus of the model legume, Medicago truncatula, providing a novel pathosystem for studying fungal-plant interactions. Data summaryAll RNA-seq data were submitted to the NCBI GEO portal under the GEO accession GSE246592. C. destructivum genome assembly and annotation are available under the NCBI BioProject PRJNA1029933 with sequence accessions CP137305-CP137317. Supplementary data (genomic and annotation files, genome browser) are available from the INRAE BIOGER Bioinformatics platform (https://bioinfo.bioger.inrae.fr/). Transposable Elements consensus sequences are also available from the French national data repository, research.data.gouv.fr with doi 10.57745/TOO1JS.

genomics↗

bioRxiv2023

Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in Saccharomyces cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for an halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.

microbiology↗

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

Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

cell biology↗

bioRxiv2019

Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterised by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters (BGCs), and their expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in repression of some BGCs through H3K4 trimethylation, allowed overproduction of 3 families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate (MeJA), an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited MeJA-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive JA-Ile synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally-related molecules, suppressed JA-Ile signalling by preventing degradation of JAZ proteins, the repressors of JA responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced IAA-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.\n\nHighlightA diterpene secondary metabolite produced by a fungal pathogen suppresses plant jasmonate defense signalling by preventing the proteasomal degradation of JAZ repressor proteins.

plant biology↗