Identification of Mycoparasitism-Related Genes against the Phytopathogen Botrytis cinerea via Transcriptome Analysis of Trichoderma harzianum T4

Abstract

Trichoderma harzianum is a well-known biological control agent (BCA) that is effective against a variety of plant pathogens. In previous studies, we found that T. harzianum T4 could effectively control the gray mold in tomatoes caused by Botrytis cinerea. However, the research on its biocontrol mechanism is not comprehensive, particularly regarding the mechanism of mycoparasitism. In this study, in order to further investigate the mycoparasitism mechanism of T. harzianum T4, transcriptomic sequencing and real-time fluorescence quantitative PCR (RT-qPCR) were used to identify the differentially expressed genes (DEGs) of T. harzianum T4 at 12, 24, 48 and 72 h of growth in the cell wall of B. cinerea (BCCW) or a sucrose medium. A total of 2871 DEGs and 2148 novel genes were detected using transcriptome sequencing. Through GO and KEGG enrichment analysis, we identified genes associated with mycoparasitism at specific time periods, such as encoding kinases, signal transduction proteins, carbohydrate active enzymes, hydrolytic enzymes, transporters, antioxidant enzymes, secondary metabolite synthesis, resistance proteins, detoxification genes and genes associated with extended hyphal longevity. To validate the transcriptome data, RT-qCPR was performed on the transcriptome samples. The RT-qPCR results show that the expression trend of the genes was consistent with the RNA-Seq data. In order to validate the screened genes associated with mycoparasitism, we performed a dual-culture antagonism test on T. harzianum and B. cinerea. The results of the dual-culture RT-qPCR showed that 15 of the 24 genes were upregulated during and after contact between T. harzianum T4 and B. cinerea (the same as BCCW), which further confirmed that these genes were involved in the mycoparasitism of T. harzianum T4. In conclusion, the transcriptome data provided in this study will not only improve the annotation information of gene models in T. harzianum T4 genome, but also provide important transcriptome information regarding the process of mycoparasitism at specific time periods, which can help us to further understand the mechanism of mycoparasitism, thus providing a potential molecular target for T. harzianum T4 as a biological control agent.

Keywords: Trichoderma harzianum T4; antioxidant enzymes; carbohydrate active enzymes; hydrolytic enzymes; mycoparasitism; signal transduction proteins; transcriptome; transporters.

Figure 1

Statistical analysis of DEGs in T. harzianum T4 at different growth stages (12, 24, 48 and 72 h) in the presence of BCCW. (A) Venn diagram of DEGs. (B) Numbers of upregulated and downregulated DEGs.

Figure 2

GO functional categories of all DEGs in T. harzianum T4 grown for (A) 12 h, (B) 24 h, (C) 48 h and (D) 72 h in the presence of BCCW.

Figure 3

GO enrichment analysis bubble diagram showing all upregulated DEGs in T. harzianum T4 grown for 12 h (A), 24 h (B), 48 h (C), and 72 h (D) in the presence of BCCW. The ordinate represents the GO classification description, and the abscissa rich factor represents the ratio of differentially expressed genes of this term to all genes that were annotated in the term.

Figure 4

Bubble diagram of KEGG pathways of upregulated DEGs in T. harzianum T4 grown for (A) 12 h, (B) 24 h, (C) 48 h and (D) 72 h in the presence of BCCW.

Figure 5

Heatmap of the DEGs involved in (A) signal transduction and kinase activity; (B) carbohydrate activity enzymes; (C) transmembrane transport and transporter; and (D) antioxidant proteins in T. harzianum T4 samples treated with BCCW at different time points (12, 24, 48 and 72 h).

Figure 6

(A) Summary of DEGs of CAZymes in T. harzianum T4 grown for 12, 24, 48 and 72 h in the presence of BCCW. (B) Summary of upregulated and downregulated DEGs of CAZymes. GH, AA, CBM, CE, GT and PL represent glycoside hydrolase, auxiliary activity, carbohydrate-binding module, carbohydrate esterase, glycosyltransferase and polysaccharide lyase, respectively.

Figure 7

Comparison of RNA-Seq and real-time RT-PCR analysis. The relative expression levels of 12 single genes of T. harzianum T4 when treated with BCCW for different times (12, 24, 48 and 72 h) were compared with RNA-Seq in pairs. The histograms represent the relative expression level (log₂FC) assessed by RNA-Seq. The black dotted lines indicate the relative expression level (2^−ΔΔCt). The error bars represent the standard deviation of three duplicates.

Figure 8

Differential expression and quantitative analysis of 24 genes related to mycoparasitism from the dual-culture direct confrontation experiment. BC: before contact, C: contact, AC: after contact. (A) Expression analysis of guanine nucleotide-binding protein gamma subunit 1, HAL protein kinase, phosphorelay intermediate protein, 6-phosphofructo-2-kinase, hexokinase and glutathione S-transferase. (B) Expression analysis of glucan endo-1,3-beta-glucosidase, murein transglycosylase, beta-glucosidase, glucan endo-1,3-beta-glucosidase, exo-beta-1,3-glucanase and endochitinase. (C) Expression analysis of nitrilase, lysine-specific histone demethylase 1, peptide chain release factor 1, acid phosphatase, peroxidase and lutamate/leucine/phenylalanine/valine dehydrogenase. (D) Expression analysis of glutamate synthase precursor, mannitol-1-phosphate dehydrogenase, trehalose 6-phosphate synthase, delta-aminolevulinic acid dehydratase, pyruvate decarboxylase and leucine-2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.