The dark septate endophyte Phialocephala sphaeroides suppresses conifer pathogen transcripts and promotes root growth of Norway spruce

Abstract

Plant-associated microbes including dark septate endophytes (DSEs) of forest trees play diverse functional roles in host fitness including growth promotion and increased defence. However, little is known about the impact on the fungal transcriptome and metabolites during tripartite interaction involving plant host, endophyte and pathogen. To understand the transcriptional regulation of endophyte and pathogen during co-infection, Norway spruce (Picea abies) seedlings were infected with DSE Phialocephala sphaeroides, or conifer root-rot pathogen Heterobasidion parviporum, or both. Phialocephala sphaeroides showed low but stable transcripts abundance (a decrease of 40%) during interaction with Norway spruce and conifer pathogen. By contrast, H. parviporum transcripts were significantly reduced (92%) during co-infection. With RNA sequencing analysis, P. sphaeroides experienced a shift from cell growth to anti-stress and antagonistic responses, while it repressed the ability of H. parviporum to access carbohydrate nutrients by suppressing its carbohydrate/polysaccharide-degrading enzyme machinery. The pathogen on the other hand secreted cysteine peptidase to restrict free growth of P. sphaeroides. The expression of both DSE P. sphaeroides and pathogen H. parviporum genes encoding plant growth promotion products were equally detected in both dual and tripartite interaction systems. This was further supported by the presence of tryptophan-dependent indolic compound in liquid culture of P. sphaeroides. Norway spruce and Arabidopsis seedlings treated with P. sphaeroides culture filtrate exhibited auxin-like phenotypes, such as enhanced root hairs, and primary root elongation at low concentration but shortened primary root at high concentration. The results suggested that the presence of the endophyte had strong repressive or suppressive effect on H. parviporum transcripts encoding genes involved in nutrient acquisition.

Keywords: dark septate endophyte; fungi–fungi–plant interaction; growth promotion; transcriptome.

Figure 1.

Phenotyping of co-cultivation systems and fungal relative transcription abundance. (A) Norway spruce seedlings were cultured in autoclaved soil with inoculation of pathogen Hp, endophyte Ps and Ps + Hp. Seedlings were kept at growth chamber with 16 h/8 h light/dark at 20 °C. Photos were taken 2 months post inoculation. (B) Relative transcription abundance of Ps from PaPs and PaPsHp samples, as well as of Hp from PaHp and PaPsHp samples. Transcripts were counted as the total transcripts that mapped to the reference genome of Phialocephala scopiformis DAOMC 229536 (LKNI00000000) or Heterobasidion parviporum (assembly ASM299478v1, PDUQ00000000). Pa, Picea abies; Ps, Phialocephala sphaeroides; Hp, Heterobasidion parviporum. ^*P < 0.05; ^**P < 0.01.

Figure 2.

RNAseq profiling of Phialocephala sphaeroides in PaPs and PaPsHp samples. (A) PCA plot of total uniquely mapped reads of endophyte P. sphaeroides from PaPs and PaPsHp samples. (B) Heatmap of hierarchical clustering of uniquely mapped reads from PaPs and PaPsHp samples. (C) GO enrichment of endophyte P. sphaeroides (Ps) from samples PaPsHp and PaPs. List of differential expressed gene (DESeq2 method) was used as input for clusterProfiler 4.0 in R with P-value cutoff 0.01 and P adjust method BH. (D) KEGG pathway enrichment of P. sphaeroides upregulated genes (DESeq2 method) in PaPs and PaPsHp samples (adjusted P-value < 0.05, genecount > 3). (E) Heatmap of selected differential expressed genes (DESeq2 method) related to cell growth, stress response and antifungal activity. ^*: It was annotated as Ca-dependent kinases, which is a mistake from computational annotation. Pa, Picea abies; Ps, Phialocephala sphaeroides; Hp, Heterobasidion parviporum.

Figure 3.

Differential expressed genes of Heterobasidion parviporum. (A) PCA plot of total uniquely mapped reads of pathogen H. parviporum from PaHp and PaPsHp samples. (B) Heatmap of hierarchical clustering of uniquely mapped reads from PaHp and PaPsHp samples. Sample PaPsHp3 was not included in PCA and hierarchical clustering because of extremely low mapping rate. (C) Heatmap of annotated differential expressed genes from PaHp and PaPsHp samples. Default adjusted P-value 0.1 was used as cutoff in DESeq2 analysis. Pa, Picea abies; Ps, Phialocephala sphaeroides; Hp, Heterobasidion parviporum.

Figure 4.

Heatmap of the expression of secreted protein genes of Heterobasidion parviporum. The expression of predicted Hp effector genes and total read counts were taken into heatmap production with DESeq2 package. Raw count of transcripts with less than (≤) 5 in more than (≥) 80% samples were removed. Read counts were normalized and transformed with variance stabilizing transformation (vst) method. Genes with lower expression in PaPsHp sample were indicated in red rectangle, and genes with high expression in both PaHp and PaPsHp samples were indicated in blue rectangle. Pa, Picea abies; Ps, Phialocephala sphaeroides; Hp, Heterobasidion parviporum.

Figure 5.

Indolic compounds production and activity by Phialocephala sphaeroides. (A) The indolic compounds in P. sphaeroides liquid culture were quantified using Salkowski reagent at wavelength 530 nm. A linear model of IAA concentration and OD530 was built as standard for quantification. Two types of growth media (ME, ME + tryptophan) and two time-points (9 day, 18 day) were selected. ME, malt extract; Trp, tryptophan. T-test and plotting were performed in R (^***<0.001). (B) Root hair enrichment of 1-week-old Arabidopsis thaliana when treated with filtered filtrate of Ps liquid culture (10× dilution). Arabidopsis root tips treated with Ps medium (ME + tryptophan) was applied as negative control, and Arabidopsis root tips treated with 2 μM IAA as positive control. (C) Root elongation of 2-week-old Norway spruce seedlings when treated with filtered filtrate of Ps liquid culture (10× dilution). Seedlings treated with Ps medium (ME + tryptophan) was applied as control. Ps, Phialocephala sphaeroides.