Osmotic Stress Responses, Cell Wall Integrity, and Conidiation Are Regulated by a Histidine Kinase Sensor in Trichoderma atroviride

Abstract

Trichoderma atroviride responds to various environmental stressors through the mitogen-activated protein kinase (MAPK) Tmk3 and MAPK-kinase Pbs2 signaling pathways. In fungi, orthologues to Tmk3 are regulated by a histidine kinase (HK) sensor. However, the role of T. atroviride HKs remains unknown. In this regard, the function of the T. atroviride HK Nik1 was analyzed in response to stressors regulated by Tmk3. The growth of the Δnik1 mutant strains was compromised under hyperosmotic stress; mycelia were less resistant to lysing enzymes than the WT strain, while conidia of Δnik1 were more sensitive to Congo red; however, ∆pbs2 and ∆tmk3 strains showed a more drastic defect in cell wall stability. Light-regulated blu1 and grg2 gene expression was induced upon an osmotic shock through Pbs2-Tmk3 but was independent of Nik1. The encoding chitin synthases chs1 and chs2 genes were downregulated after an osmotic shock in the WT, but chs1 and chs3 expression were enhanced in ∆nik1, ∆pbs2, and ∆tmk3. The vegetative growth and conidiation by light decreased in ∆nik1, although Nik1 was unrequired to activate the light-responsive genes by Tmk3. Altogether, Nik1 regulates responses related to the Pbs2-Tmk3 pathway and suggests the participation of additional HKs to respond to stress.

Keywords: MAPK signaling; cell wall integrity; histidine kinase; sporulation; stress cellular.

Figure 1

Nik1 amino acid sequence analysis. (A) Domains of the T. atroviride Nik1 protein predicted by SMART [27]. HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; HisKA, phosphoacceptor domain; HATPase_c, ATP-binding domain; REC, receiver domain. (B,C) Amino acid sequence alignment to identify the conserved phosphorylable His⁷²⁰ residue in the HisKA motif (B) and the Asp¹¹³⁹ residue in the REC domain (C). Alignment was carried out using MegAlign software version 7.1.0 (DNAStar) by the Clustal W Method. Ta, T. atroviride IMI206040; Nc, N. crassa; Pg, P. grisea; An, A. nidulans; Ca, C. albicans.

Figure 2

The HK Nik1 regulates tolerance to high osmolarity challenges. (A) Tolerance to osmotic stress in the conidia of WT, ∆nik1, ∆pbs2, and ∆tmk3 strains. Drops of 500 conidia of the WT and mutant strains were inoculated on PDA plates plus different concentrations of the indicated stressors. Plates were incubated at 27 °C for four days. (B) Tolerance to osmotic stress in the mycelia of WT, ∆nik1, ∆pbs2, and ∆tmk3 strains. NaCl and sorbitol were added to PDA media at the concentrations indicated. Strains were incubated at 27 °C for four days. All experiments were carried out in triplicate.

Figure 3

The HK Nik1 plays a role in cell wall integrity. (A) Tolerance to Congo red in the conidia of WT, ∆nik1, ∆pbs2, and ∆tmk3 strains. Drops of 500 conidia of the WT and mutant strains were inoculated on PDA plates with Congo red at the indicated concentrations, incubated at 27 °C for four days in constant white light and darkness, and pictures were taken. The strain order is indicated in Figure 2. (B) Sensitivity to cell wall lysing enzymes of WT, ∆nik1, ∆pbs2, and ∆tmk3 strains. The total production of protoplasts was determined using a Neubauer chamber. The mean value is represented in bars ± SEM of three independent experiments analyzed with the Tukey–Kramer method (α = 0.001). * and ** indicate mean values that are statistically different from the control.

Figure 4

Expression analysis in response to osmotic stress. (A). The mycelia of the WT strain were challenged by adding 0.5 M NaCl. Then, samples were collected at the indicated time. Total RNA was extracted from samples and used to synthesize cDNA. As a loading control, the gpd gene was amplified. In the same reaction for cDNA synthesis, the RNA of samples without transcriptase was used as a template to amplify gpd as a negative control. (B,C) Semiquantitative transcript levels were determined according to signal intensity from two biological replicates and plotted. The expression level for each gene was normalized by dividing the control gpd signal. Relative expression was adjusted to the unit in the control (0 min) without an osmotic shock and compared with the treatment at different time points.

Figure 5

Role of Nik1, Pbs2, and Tmk3 on gene expression regulated by osmotic stress. (A) Total RNA was extracted from the mycelia of WT and mutant strains that were non-stressed (−) or 15 min after an osmotic shock by 0.5 M NaCl (+) and used to synthesize cDNA. In the same reaction for cDNA synthesis, (1) RNA of samples without transcriptase was used as a negative control and (2) gpd was used as a loading control (cDNA template). (B) Signal intensity was quantified from two biological replicates and plotted as indicated in Figure 4. The expression level for each gene was normalized and divided by the control gpd signal. Relative expression was adjusted to the unit in the WT without osmotic stress (−).

Figure 6

Asexual reproduction and gene expression regulated by light in ∆nik1 mutants. (A) Conidial production of the WT strain and the ∆nik1, ∆pbs2, and ∆tmk3 mutant strains. Mycelial plugs of the indicated strains were inoculated on PDA plates, incubated at 27 °C in darkness for 36 h, and then exposed to a blue light pulse (152.4 μmol m⁻²) (LP), or grown for 7 days in constant white light (0.586 μmol m⁻² s⁻¹) (CL). The mean value of conidia yield is represented in bars ± SEM of three independent experiments analyzed with the Tukey–Kramer method (α = 0.05). * and ** indicate mean values that are statistically different from the control. (B) Expression analysis by RT-PCR of light-induced genes in the WT, ∆pbs2, ∆tmk3, and ∆nik1 strains. D, darkness; L, light.