Proteomics of Two Thermotolerant Isolates of Trichoderma under High-Temperature Stress

Abstract

Several species of the soil borne fungus of the genus Trichoderma are known to be versatile, opportunistic plant symbionts and are the most successful biocontrol agents used in today's agriculture. To be successful in field conditions, the fungus must endure varying climatic conditions. Studies have indicated that a high atmospheric temperature coupled with low humidity is a major factor in the inconsistent performance of Trichoderma under field conditions. Understanding the molecular modulations associated with Trichoderma that persist and deliver under abiotic stress conditions will aid in exploiting the value of these organisms for such uses. In this study, a comparative proteomic analysis, using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/time-of-flight (MALDI-TOF-TOF) mass spectrometry, was used to identify proteins associated with thermotolerance in two thermotolerant isolates of Trichoderma: T. longibrachiatum 673, TaDOR673 and T. asperellum 7316, TaDOR7316; with 32 differentially expressed proteins being identified. Sequence homology and conserved domains were used to identify these proteins and to assign a probable function to them. The thermotolerant isolate, TaDOR673, seemed to employ the stress signaling MAPK pathways and heat shock response pathways to combat the stress condition, whereas the moderately tolerant isolate, TaDOR7316, seemed to adapt to high-temperature conditions by reducing the accumulation of misfolded proteins through an unfolded protein response pathway and autophagy. In addition, there were unique, as well as common, proteins that were differentially expressed in the two isolates studied.

Keywords: Hsf1; MAPK; Trichoderma; UPR; autophagy; cell wall remodeling; thermotolerance.

Figure 1

Morphology of thermotolerant isolates of Trichoderma.

Figure 2

Two-dimensional gel image of the protein expression pattern in T. longibrachiatum 673, TaDOR673 under (a) control (grown at 28 °C) and heat stress conditions at 48 °C for 1 h (b) and 4 h (c). The gels were obtained in duplicates; a representative of each duplicate is shown. Identified protein spots are numbered and listed in Table 1. The pH gradient is marked above the gel, and the molecular mass protein standards (kDa) are indicated on the left of the gel.

Figure 3

Two-dimensional gel image of the protein expression pattern in T. asperellum 7316, TaDOR7316 under (a) control (grown at 28 °C) and heat stress conditions at 48 °C for 1 h (b) and 4 h (c). The gels were obtained in duplicates; a representative of each duplicate is shown. Identified protein spots are numbered and listed in Table 2. The pH gradient is marked above the gel, and the molecular mass protein standards (kDa) are indicated on the left of the gel.

Figure 4

3D view of differential intensity levels of some of the protein spots identified in thermotolerant isolates of Trichoderma during the heat stress. Spot intensity was quantified by Image Master 2-D Platinum V6.0 image analysis software (GE Healthcare). The images show a peak for each protein spot, with a peak height that is proportional to the spot intensity. (C: control; 1 h: Heat stress at 48 °C for 1 h; 4 h: Heat stress at 48 °C for 4 h).