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Schematic representation of the 77 secondary metabolism gene clusters of C. higginsianum. (PDF 374 kb)

Schematic representation of the association between four segmental duplications (shown in green) with the C. higginsianum subtelomeric repeats DHX_G198, DTX_chim-G199, and DHX_chim-G203. Homologous regions are shaded grey-blue. (PDF 747 kb)

Stage-specific expression of C. higginsianum transposable elements (TEs). (A) Heatmap showing the expression profiles of TEs. (B) K-means clustering of 441 TE copies considered to be expressed in at least one fungal stage (VA = in vitro appressoria, PA = in planta appressoria, BP = biotrophic phase). For each of the five clusters, the average profile is shown in red. (C) Localization of in planta-expressed LTR transposon fragments in the 3’ UTR regions of C. higginsianum effector genes. IGV screenshots showing the genomic locations of TE copies RXX_R113 and RLX_P25.13 (red) in relation to effector genes ChEC35 and ChEC117 (green), respectively. RNA-Seq reads are displayed for appressoria in vitro (VA) and the biotrophic phase (BP). The RLX_P25.13 copy comprises a ‘solo’-LTR, likely produced by homologous recombination between two LTRs, leading to deletion of the internal retrotransposon sequence. (PDF 2850 kb)

Summary of unitigs comprising the C. higginsianum assembly. (PDF 91 kb)

(A) Dinucleotide mutation bias among TE copies belonging to different TE orders. Mutation rates were calculated using RIPCAL by comparing each TE copy with a Ti/Tv > 1.5 to the TE consensus sequence. Y-axis: percentage relative to the total number of copies used in RIPCAL analysis. Coloured bars indicate the percentage of copies with expected RIP (Ti/Tv > 1.5) and dinucleotide preferentially used (>1/3) in CN- > TN and (cNG - > cNA) mutations. Black bar: percentage of copies without expected RIP (Ti/Tv > 1.5). Gray bar: percentage of copies with expected RIP (Ti/Tv > 1.5) but no evidence of dinucleotide bias. (B) Plot showing the sequence divergence of TE copies belonging to different TE orders relative to their respective consensus sequences. (PDF 466 kb)

(A) PCR products encompassing the two break-points shown in Fig. 1 confirm the unitig sequences rather than the optical maps. PCR validation of putative break-points using primer pairs P812 - P813 and P422 - P423. If the assembly is correct, the expected PCR products are respectively 3992 bp and 5470 bp long. L: Generuler 1 kb Plus DNA Ladder; 1: unitig_7 break-point; 2: unitig_9 break-point. (B) PCR validation of segmental duplication SD2. Primers P677, P678 and P679 are colour-coded for the features they match. L: Generuler 1 kb Plus DNA Ladder; 3: P678xP677 amplicon; 4: P679xP677 amplicon. (PDF 2434 kb)

List of the secondary metabolism key genes and their catalytic domains. (PDF 132 kb)

Characteristics and contents of the 12 largest unitigs in the new genome assembly, corresponding to the 12 chromosomes of C. higginsianum. (PDF 129 kb)

Colletotrichum higginsianum is an ascomycete fungus causing anthracnose disease on numerous cultivated plants in the familyBrassicaceae, as well as the model plant Arabidopsis thaliana. We report an assembly of the nuclear genome and gene annotation of this pathogen, which was obtained using a combination of PacBio long-read sequencing and optical mapping

Species of the genus Colletotrichum cause devastating anthracnose or blight diseases on numerous crop plants worldwide. C. higginsianum uses a hemibiotrophic strategy to infect Arabidopsis and other Brassicaceae. Compared to other hemibiotrophs, its genome contains an exceptionally large number of genes (78) encoding secondary metabolism (SM) key enzymes. As in other fungi, these key genes are organized into clusters that also contain genes encoding accessory enzymes of the same biosynthetic pathway and usually efflux transporters and pathway-specific transcription factors. A remarkable finding from RNA-Seq transcriptome profiling was that 27 SM clusters are specifically expressed only in planta by appressoria and/or biotrophic hyphae. Since each cluster potentially synthesizes one final metabolite, this suggests appressoria and biotrophic hyphae deliver a cocktail of different metabolites to the first infected host cell. At this early stage host cells remain alive, raising the possibility that these molecules function similar to protein effectors in host manipulation, rather than as simple phytotoxins. Work is ongoing to decipher how the expression of these SM gene clusters is so precisely regulated during the course of infection. We targeted C. higginsianum orthologs of the well-characterized global SM regulators LaeA, Hp1, Dim5, CclA and Kmt6 for genetic manipulation, with a view to inducing the fungus to synthesize appressorial metabolites in large quantities in culture. The aim is to obtain sufficient material for identification and structural determination (mass spectrometry, NMR) and for functional assays to evaluate their biological activities against plants, bacteria and fungi.