Survival trade-offs in plant roots during colonization by closely related beneficial and pathogenic fungi

Abstract

The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.

Figure 1. Colletotrichum evolutionary divergence dates and SNP distribution in C. tofieldiae isolates.

(a) Phylogeny of Colletotrichum species inferred from analysing 20 single-copy gene families using PhyML and r8s. Nodes 1–3 (green) are calibration points and nodes 4, 5 and 6 (red) represent estimated divergence dates (see Supplementary Note 3). (b) Circular visualization of the alignment of genome sequencing reads and SNP locations of four C. tofieldiae isolates with respect to the Ct0861 reference assembly. Tracks represent (from the outside) the five largest Ct0861 contigs (scale: kb); locations of predicted genes; locations of SNPs versus Ct0861 in CBS495, CBS130, CBS127, CBS168 (see Supplementary Table 1 for full culture IDs) and SNPs common to these four isolates; conserved regions with low SNP density between all the five isolates; mean read coverage (per 100 bases) for isolates CBS495, CBS130, CBS127 and CBS168. Coverage plot scales are 0 to 1,000 (CBS495) or 0 to 500 (CBS130, 127, 168). (c) SNP density (per 1 kb) in isolates CBS495, CBS130, CBS127 and CBS168 versus Ct0861, compared with gene density (per 10 kb) and GC content (%) on the five largest Ct0861 contigs.

Figure 2. Conservation of orthoMCL gene families within the proteomes of Colletotrichum species.

(a) Heatmap and hierarchical clustering dendrogram depicting the percentage of gene families shared between 10 Colletotrichum genomes. Node labels in the tree indicate bootstrap support after 100 iterations. Brackets (right-hand side) indicate the number of gene families shared between the groups of genomes. (b) Upper panel: Venn diagram of gene families shared between the beneficial C. tofieldiae 0861 and its close pathogenic relative, C. incanum. Lower panel: Barplot showing the over-abundance of proteins related to secondary metabolite biosynthesis among gene families unique to Ct0861 compared with all C. tofieldiae gene families (Fisher's exact test; ***P=3.31E−08).

Figure 3. Conservation and expression of genes encoding candidate secreted effector proteins in C. tofieldiae and C. incanum.

(a) Proportions of predicted secreted proteins (circles, violet sectors) and candidate secreted effector proteins CSEPs (circles, yellow sectors) in the proteomes and secretomes of Colletotrichum species, respectively. The number of genus- and species-specific CSEPs detected for each species is indicated in the barplot. (b) Boxplot with a rotated kernel density on each side showing d_N/d_S ratio (log₁₀) measured in the proteome, the secretome and the CSEP repertoires of 10 Colletotrichum isolates using the gene families defined by MCL clustering (see Fig. 2). The overall d_N/d_S ratio is significantly higher for gene families encoding secreted proteins and CSEPs compared with the remaining gene families (One-sided Fisher's test, ***P<0.001). (c,d) Expression and regulation of CSEPs in C. tofieldiae 0861 (c) and C. incanum (d). The circular plots show (from the inside): dendrograms of the CSEPs based on protein sequence alignments, CSEP length (0–500 amino acids), species-specific (Sp, black) and genus-specific (Ge, white) CSEPs, normalized gene expression (Exp.) levels in vitro (IV) and in planta (IP) at 10 and 24 days post inoculation, CSEPs significantly up- (violet) and downregulated (green) at 10 days post inoculation versus in vitro (10IP/IV) and 24 days post inoculation versus in vitro (24IP/IV) (|log₂FC|⩾1, FDR<0.05).

Figure 4. Colletotrichum CAZyme repertoires and their transcriptional regulation in C. tofieldiae and C. incanum.

(a) Hierarchical clustering of CAZyme classes from the genomes of four Colletotrichum species. AA, auxiliary activities; CBM, carbohydrate-binding module CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyltransferase; PL, polysaccharide lyase. The numbers of enzyme modules in each genome are shown. Overrepresented (dark grey to black) and underrepresented (pale grey to white) modules are depicted as log₂ (fold changes) relative to the class mean. (b) Transcript profiling of C. tofieldiae CAZyme genes in vitro and during colonization of Arabidopsis roots at 6, 10, 16 and 24 days post inoculation (d.p.i.) under phosphate sufficient (+P: [625 μM]) and deficient (−P: [50 μM]) conditions. (c) Transcript profiling of C. incanum CAZyme genes in vitro and during colonization of Arabidopsis roots at 10 and 24 d.p.i. under phosphate-deficient conditions (−P: [50 μM]). (b,c) Overrepresented (yellow to red) and underrepresented transcripts (yellow to blue) are shown as log₂ (fold changes) relative to the mean expression across all the stages. The red marks represent secreted CAZymes and the black marks indicate involvement in metabolic activities linked to energy storage and exchange (Energy), or degradation of fungal cell walls (FCW), plant cell walls (PCW) or both (F or PCW). For CAZymes acting on PCW, the corresponding plant substrates (cellulose, hemicellulose, hemicellulose and pectin, pectin) are indicated by a colour code. REI, relative expression index.

Figure 5. Transcriptional reprogramming of Pi-starved and non-starved Arabidopsis roots in response to C. tofieldiae.

(a) Transcript profiling of 5,561 Arabidopsis genes significantly regulated (moderated t-test, |log₂FC|⩾1, FDR<0.05) between colonized versus mock-treated roots and phosphate-starved (−P: [50 μM]) versus non-starved roots (+P: [625 μM]) at 6, 10, 16 and 24 days post inoculation. Overrepresented (yellow to red) and underrepresented transcripts (yellow to blue) are shown as log₂ (fold changes) relative to the mean expression across all stages. Using k-means partitioning, the gene set was split into 20 major gene expression clusters. (b) Gene Ontology term enrichment network analysis among the 10 clusters highlighted in a. Each significantly enriched GO term (P<0.05, hypergeometric test, Bonferroni step-down correction) is represented with a circle and the contribution (%) of each cluster to the overall GO term enrichment is represented using the same colour code as in a. As tightly connected GO terms are functionally linked, only the major host responses outputs are indicated (dotted line). (c) For cluster 8 and cluster 9, gene relationships based on co-regulation were assessed using other Arabidopsis expression data sets (see Supplementary Note 9). The genes within each cluster that show strong expression relationships in other expression data sets are likely to encode key regulatory hubs. Hub genes (cluster 9: ⩾5 connections, *cluster 8: ⩾10 connections) are highlighted in black. The corresponding characterized Arabidopsis genes are indicated below the co-expression networks. (d) Validation of the expression profiles of the hub genes RHS19 (cluster 8) and ERF13 (cluster 9) using RT–qPCR (see Supplementary Note 10). Error bars indicate standard error (n=3 biological replicates), NA, data not available; REI, Relative Expression Index.

Figure 6. Comparative transcriptome analysis of Arabidopsis roots in response to beneficial C. tofieldiae and pathogenic C. incanum.

(a) Transcript profiling of 2,009 Arabidopsis genes significantly regulated (moderated t-test, |log₂FC|⩾1, FDR<0.05) between C. incanum- versus (vs) C. tofieldiae-colonized roots at 10 days post inoculation (d.p.i.) under phosphate-deficient conditions (−P: 50 μM). Overrepresented (yellow to red) and underrepresented transcripts (yellow to blue) are shown as log₂ (fold changes) relative to the mean expression across all stages. E1 and E2 correspond to two fully independent experiments (see Supplementary Note 9). Gene expression fold changes (green: downregulated; violet: upregulated) were calculated between C. tofieldiae-colonized versus mock-treated roots, C. incanum-colonized versus mock-treated roots or C. incanum-colonized versus C. tofieldiae-colonized roots. (b) GO term enrichment analysis of Arabidopsis genes preferentially expressed in response to C. tofieldiae (Cluster 1) or in response to C. incanum (cluster 2). Each circle corresponds to a significantly enriched GO term (P<0.05, hypergeometric test, Bonferroni step-down correction). The colour code reflects P values and the circle size the number of genes associated to each GO term. Similar to Fig. 5b, the GO terms that are tightly connected are functionally linked and therefore only the major host-response outputs are indicated (dotted line). REI, Relative Expression Index.