Genome-wide transcriptome profiling reveals molecular response pathways of Trichoderma harzianum in response to salt stress

Abstract

Trichoderma harzianum exhibits a strong biological control effect on many important plant pathogens, such as Fusarium oxysporum, Botrytis cinerea, and Meloidogyne. However, its biocontrol effectiveness is weakened or reduced under salt stress. The aim of this study was to investigate the molecular response of T. harzianum to salt stress at the whole-genome level. Here, we present a 44.47 Mb near-complete genome assembly of the T. harzianum qt40003 strain for the first time, which was assembled de novo with 7.59 Gb Nanopore sequencing long reads (~170-fold) and 5.2 Gb Illumina short reads (~116-fold). The assembled qt40003 genome contains 12 contigs, with a contig N50 of 4.81 Mb, in which four of the 12 contigs were entirely reconstructed in a single chromosome from telomere to telomere. The qt40003 genome contains 4.27 Mb of repeat sequences and 12,238 protein-coding genes with a BUSCO completeness of 97.5%, indicating the high accuracy and completeness of our gene annotations. Genome-wide transcriptomic analysis was used to investigate gene expression changes related to salt stress in qt40003 at 0, 2% (T2), and 4% (T4) sodium chloride concentrations. A total of 2,937 and 3,527 differentially expressed genes (DEGs) were obtained under T2 and T4 conditions, respectively. GO enrichment analysis showed that the T2-treatment DEGs were highly enriched in detoxification (p < 0.001), while the T4 DEGs were mainly enriched in cell components, mostly in cellular detoxification, cell surface, and cell wall. KEGG metabolic pathway analysis showed that 91 and 173 DEGs were significantly enriched in the T2 and T4 treatments, respectively (p < 0.01), mainly in the glutathione metabolism pathway. We further experimentally analyzed the differentially expressed glutathione transferase genes in the glutathione metabolic pathway, most of which were downregulated (13/15). In addition, we screened 13 genes related to active oxygen clearance, including six upregulated and seven downregulated genes, alongside five fungal hydrophobic proteins, of which two genes were highly expressed. Our study provides high-quality genome information for the use of T. harzianum for biological control and offers significant insights into the molecular responses of T. harzianum under salt-stress conditions.

Keywords: NaCl; genome; metabolic; salt tolerance mechanism; transcriptomic.

Figure 1

The morphological and molecular identification of qt40003. (A) The colony morphology of qt40003. (B) Microscopic observation of conidiophore. (C,D) Microscopic observation of mycelia and conidia. (E) PCR molecular identification was performed with specific primers. (Marker: Trans 2Kb DNA Marker, qt40001 and qt40443: other Trichoderma harzianum strains, TEF: Translation Elongation Factor and RPB2: RNA Polymerase II Second Largest Subunit). (F) Phylogenetic tree based on RPB2 sequences of qt40003 and 8 other different Trichoderma species.

Figure 2

Genome features of qt40003 and comparative genomic analysis with published T. harzianum genome. (A) The outermost circle is the contigs. (B) The syntenic relationships between qt40003 and IIPRTh-33. Y-axis represents qt40003 contigs, and x-axis represents IIPRTh-33 genome. The axis tick values represent genome size (×10 Mb). The purple dot or line represents forward matches, and the blue dot or line represents reverse matches between two genomes.

Figure 3

Effects of different concentrations of NaCl on the growth of qt40003. (A) 48 h growth of strain qt40003 on NaCl PDA. (B) Colony size at different NaCl concentrations. (C) Inhibition rate of different NaCl concentrations on the growth of strains. Error lines are standard errors.

Figure 4

Go enrichment classification of differentially expressed genes under different treatments. (A) Go enrichment classification of differentially expressed genes treated with T2. (B) Go enrichment classification of differentially expressed genes treated with T4.

Figure 5

KEGG enrichment classification of differentially expressed genes under different treatments. (A) KEGG enrichment classification of differentially expressed genes treated with T2. (B) KEGG enrichment classification of differentially expressed genes treated with T4.

Figure 6

Relationship diagram of differentially expressed genes in the glutathione metabolic pathway. (A) Heat map of differential expression genes of glutathione metabolic pathways. (B) Phylogenetic tree of glutathione transferase gene family showing its evolution. Bootstrap values (n = 1,000).

Figure 7

Analysis of non-overlapping differentially expressed genes across distinct treatments. (A) Venn diagram of differentially expressed genes under T2 and T4 treatments. (B) KEGG metabolic pathway annotation of non-overlapping differentially expressed genes under different treatments.

Figure 8

qPCR validation of differentially expressed genes. The reliability of RNA-seq results for 10 differentially expressed genes was substantiated through qPCR validation. FPKM values from the RNA-seq analysis were presented in the orange column, while qPCR measurements were displayed in the green column. The data were expressed as the mean ± standard error (n = 3). In the context of an unpaired t-test, “*” signified a significance level of p < 0.05, “**” indicated p < 0.01, “***” denoted p < 0.001, “****” represented an exceptionally profound significance, and “ns” indicated a lack of statistical significance.