Transcriptome reprogramming, epigenetic modifications and alternative splicing orchestrate the tomato root response to the beneficial fungus Trichoderma harzianum

Abstract

Beneficial interactions of rhizosphere microorganisms are widely exploited for plant biofertilization and mitigation of biotic and abiotic constraints. To provide new insights into the onset of the roots-beneficial microorganisms interplay, we characterised the transcriptomes expressed in tomato roots at 24, 48 and 72 h post inoculation with the beneficial fungus Trichoderma harzianum T22 and analysed the epigenetic and post-trascriptional regulation mechanisms. We detected 1243 tomato transcripts that were differentially expressed between Trichoderma-interacting and control roots and 83 T. harzianum transcripts that were differentially expressed between the three experimental time points. Interaction with Trichoderma triggered a transcriptional response mainly ascribable to signal recognition and transduction, stress response, transcriptional regulation and transport. In tomato roots, salicylic acid, and not jasmonate, appears to have a prominent role in orchestrating the interplay with this beneficial strain. Differential regulation of many nutrient transporter genes indicated a strong effect on plant nutrition processes, which, together with the possible modifications in root architecture triggered by ethylene/indole-3-acetic acid signalling at 72 h post inoculation may concur to the well-described growth-promotion ability of this strain. Alongside, T. harzianum-induced defence priming and stress tolerance may be mediated by the induction of reactive oxygen species, detoxification and defence genes. A deeper insight into gene expression and regulation control provided first evidences for the involvement of cytosine methylation and alternative splicing mechanisms in the plant-Trichoderma interaction. A model is proposed that integrates the plant transcriptomic responses in the roots, where interaction between the plant and beneficial rhizosphere microorganisms occurs.

Fig. 1. Venn diagram of tomato's differentially expressed genes in T. harzianum T22-treated vs. control roots.

The distribution of the 1243 differentially expressed genes (DEGs) in tomato root resulting from RNA-seq analysis is reported for different times of interaction (24, 48 and 72 hpi) with the beneficial fungus T. harzianum. DEGs were identified setting the false discovery rate at 10% (p < 0.1) and the minimum fold change at ±1.1

Fig. 2. Clustering of expression profiles of tomato root genes differentially expressed during the interaction with T. harzianum T22 and gene ontology enrichment analysis.

a Grouping of tomato genes modulated by T. harzianum into five clusters, according to their expression profiles across the interaction period (24, 48 and 72 hpi), using the Pearson’s correlation distance (SOTA method). The number of genes assigned to each cluster is indicated. The thick line indicates the cluster centroid. The y-axis represents the fold change of the gene expression level. b Significantly enriched gene ontology (GO) terms associated with each expression cluster, ordered according to increasing p-value. Black and grey bars represent the biological process (BP) and molecular function (MF) categories, respectively. The x-axis represents the number of genes grouped in each GO category. TF, transcription factor

Fig. 3. MapMan analysis of tomato root genes differentially expressed during the interaction with T. harzianum T22.

Organisation of tomato root's differentially expressed genes (DEGs) in functional categories (bins) according to the MapMan ontology across the interaction period (24, 48 and 72 hpi). Genes significantly upregulated and downregulated in treated vs. control plants are indicated in red and blue, respectively. The colour set scale is on top right corner

Fig. 4. Absolute levels of global DNA methylation in control and Trichoderma-treated tomato roots.

DNA methylation was assessed across the interaction period (24, 48 and 72 hpi) and reported as percent content of 5-methylCytosine (% 5-mC) using an antibody-based colorimetric detection kit. Methylation levels significantly different from the corresponding control are indicated by *p < 0.05

Fig. 5. Venn diagrams of tomato gene loci affected by alternative splicing in control and Trichoderma-treated roots.

Distribution of novel transcribed isoforms and/or gene features (‘intron only’, ‘multiple annotation’ and ‘internal exon extension’ categories) resulting from comparison with iTAG2.4 annotations for a control and b Trichoderma-treated root samples at 24, 48 and 72 hpi. R-SAP analysis was run with IdentityCutoff = 95 and CovCutoff = 98

Fig. 6. Overview of modulated gene expression in tomato roots during T. harzianum T22 colonisation.

The illustration is based on annotation of the DEGs in iTAG2.4 and assignment to specific functional categories by MapMan ontology. Tomato genes that were significantly upregulated and downregulated in response to T. harzianum at each time point (24, 48 and 72 hpi) are in red and green, respectively. Gene families including both downregulated and upregulated members are in blue. Trichoderma transcripts involved in putative MAMP/DAMPs elicitation are also indicated. Solid and dashed lines represent established and hypothesised activities, respectively

Fig. 7. Levels of salicylic acid (SA) and salicylic acid glucoside (SAG) in tomato roots.

The levels of a SA and b SAG were determined in control and T. harzianum-treated roots across the interaction period (24, 48 and 72 hpi) by quantitative HPLC-MS/MS. Bars represent the means ± SD of three biological replicates. Data were subjected to analysis of variance and tested for significance (p < 0.05) using the Tukey’s test. FW, fresh weight

Fig. 8. Proposed model of the early events occurring in the root–Trichoderma interaction.

Recognition of T. harzianum MAMPs/DAMPs elicitors by tomato roots' pattern recognition receptors triggers MTI/DTI response across the observed interaction period (from 24 to 72 hpi). Phytohormone cross-talk orchestrates root colonisation by Trichoderma: (1) Induction of SA biosynthesis and signalling as well as ROS accumulation activate plant defence, thus limiting fungal spread and (2) SA-induced inhibition of JA and Et biosynthesis and signalling allows controlled root colonisation. At later times, increased Et and auxin signalling induce modifications of root architecture that, together with changes in nutrient transport, stimulate plant growth