Functions of the C2H2 Transcription Factor Gene thmea1 in Trichoderma harzianum under Copper Stress Based on Transcriptome Analysis

Abstract

Trichoderma spp. are important biocontrol filamentous fungi and have tremendous potential in soil bioremediation. In our previous studies, a C2H2 type transcription factor coding gene (thmea1) was cloned from a biocontrol agent T. harzianum Th-33; the encoded sequence of thmea1 contained 3 conserved C2H2 domains with Swi5 and Ace2 in Saccharomyces cerevisiae. The thmea1 knockout mutant Δthmea1 showed 12.9% higher copper tolerance than the wild-type Th33. To elucidate the function of thmea1 and its relationship with copper stress response, we conducted transcriptome sequencing and analysis of wild-type Th33 and Δthmea1 under 0.8 mM copper stress. A total of 1061 differentially expressed genes (DEGs) were identified between the two strains, all DEGs were assigned to KEGG pathway database, 383 DEGs were annotated in 191 individual pathways, and the categories of ribosomal protein synthesis and amino acid metabolism were the most highly enriched ones. Analysis of related DEGs showed that the expression levels of intracellular glutathione detoxification enzyme, heat shock proteins, and ribosomal proteins in Δthmea1 were higher than that of the wild-type Th33, and the expression of metallothionein (MT) gene did not change. In addition, the expression levels of genes coding for proteins associated with the Ccc2p-mediated copper chaperone Atx1p transport of copper ions into the Golgi secretory pathway increased, as well as the copper amine oxidase (CuAO). These findings suggest that Thmea1 is a negative regulated factor of copper tolerance ability in T. harzianum. It does not show metallothionein expression activator activities as that of Ace2 in S. cerevisiae. We hypothesize that after T. harzianum has lost its thmea1 gene, the ability of cells to scavenge reactive oxygen species, mainly through the glutathione antioxidant system, is enhanced, whereas protein synthesis and repair and copper secretion increase under copper stress, which increases the ability of the mutant strain to tolerate copper stress.

Figure 1

Effects of copper ions on the growth rate and colony morphology of Trichoderma harzianum Th33 and thmea1 gene knockout mutant Δthmea1. (a) Colony diameter of T. harzianum Th33 wild-type and mutant Δthmea1 under different copper iron concentrations at 28°C for 2 days. (b) Colony morphology of T. harzianum Th33 wild-type and mutant Δthmea1 strains under different copper concentrations at 28°C for 2 days. Note: figure data is expressed as mean ± standard error. ∗ represents significance level of differences under different treatments (∗ p<0.05; ∗∗ p<0.01).

Figure 2

Differentially expressed genes of ΔTH-0.8_VS_Th33-0.8.

Figure 3

GO enrichment analysis of differentially expressed genes.

Figure 4

Pathway enrichment analysis of DEGs. The percentage of differentially expressed genes involved in KEGG pathways. Only the top 17 most abundant KEGG pathways are represented.

Figure 5

Validation of transcriptome sequencing results. (a) The transcriptome expression levels of 12 differentially expressed genes were expressed as log₂FPKM; (b) qRT-PCR analysis of 12 differentially expressed genes. Note: figure data is expressed as the mean ± standard error.

Figure 6

Proposed copper metabolism diagram in Trichoderma harzianum.