Transcriptional responses of soybean roots to colonization with the root endophytic fungus Piriformospora indica reveals altered phenylpropanoid and secondary metabolism

Abstract

Piriformospora indica, a root endophytic fungus, has been shown to enhance biomass production and confer tolerance to various abiotic and biotic stresses in many plant hosts. A growth chamber experiment of soybean (Glycine max) colonized by P. indica compared to uninoculated control plants showed that the fungus significantly increased shoot dry weight, nutrient content, and rhizobial biomass. RNA-Seq analyses of root tissue showed upregulation of 61 genes and downregulation of 238 genes in colonized plants. Gene Ontology (GO) enrichment analyses demonstrated that upregulated genes were most significantly enriched in GO categories related to lignin biosynthesis and regulation of iron transport and metabolism but also mapped to categories of nutrient acquisition, hormone signaling, and response to drought stress. Metabolic pathway analysis revealed upregulation of genes within the phenylpropanoid and derivative pathways such as biosynthesis of monolignol subunits, flavonoids and flavonols (luteolin and quercetin), and iron scavenging siderophores. Highly enriched downregulated GO categories included heat shock proteins involved in response to heat, high-light intensity, hydrogen peroxide, and several related to plant defense. Overall, these results suggest that soybean maintains an association with this root endosymbiotic fungus that improves plant growth and nutrient acquisition, modulates abiotic stress, and promotes synergistic interactions with rhizobia.

Figure 1

(A) Roots of control and P. indica treatments as seen under 40X magnification after staining with lactophenol cotton blue. Pear-shaped chlamydospores were observed in P. indica treatment (arrows) only while no chlamydospores was observed in the control. (B) Shoots of control and P. indica colonized plants. There was no significant difference in shoot lengths but the shoot dry weights increased in P. indica treatment. (C) Roots of control and P. indica colonized plants. The root dry weight decreased in P. indica colonized plants. (D) Nodules in control and P. indica treatments. Numbers of nodules decreased while average size increased in P. indica treatment.

Figure 2

(A) Total number of genes expressed in control only (light green), P. indica treatment only (gray), and genes expressed in both conditions (dark green). (B) Graph of the number of genes upregulated and down regulated in P. indica colonized roots compared to the control at FDR = 0.05.

Figure 3

Differentially expressed genes summarized as a tree network using REVIGO. (A) GO terms related to biological processes of upregulated genes in the comparison of control versus P. indica. (B) GO terms related to biological processes of downregulated genes in the comparison control versus P. indica. GO categories are represented by circles and are color coded according to corrected p-value for GO enrichment analysis. Red indicates categories that are significantly enriched at p < 0.05 while nonsignificant categories are color coded with corrected p < 0.5 = orange, p < 1 = yellow, p < 5 = green, and p < 10 = blue). Lines indicate connections between categories within the GO hierarchy and singletons have been grouped by similar function.

Figure 4

Effect of colonization with P. indica on the expression of genes within the phenylpropanoid biosynthetic pathway. Red colored squares indicate genes upregulated in the comparison of control versus P. indica; blue colored squares indicate down-regulated genes in this comparison. Homologs in Arabidopsis of all upregulated and downregulated soybean genes were mapped to the Arabidopsis KEGG pathway for phenylpropanoid biosynthesis^–. Enzyme Commission (EC) codes colored in green boxes represent enzymatic steps present in soybean. Steps involved in biosynthesis of monolignol precursors sinapyl alcohol, coniferal alcohol, and 5-hydroxy-coniferal alcohol are shaded in the orange box. The final steps of production of G and S lignin (shaded brown box) are performed by peroxidases located within the plant cell wall. Biosynthesis of the iron-scavenging coumarin scopoletin is shaded in the pink box.

Figure 5

Effect of colonization with P. indica on the expression of genes within the flavonoid biosynthetic pathway. Red colored squares indicate genes upregulated in the comparison of control versus P. indica; blue colored squares indicate down-regulated genes in this comparison. Homologs in Arabidopsis of all upregulated and downregulated soybean genes were mapped to the Arabidopsis KEGG pathway for flavonoid biosynthesis^–. Enzyme Commission (EC) codes colored in green boxes represent enzymatic steps present in soybean. While several steps early in the pathway are downregulated, genes in steps leading to biosynthesis of luteolin (purple box) and quercetin (pink box) are upregulated.