Biotechnological applications of the gene transfer from the beneficial fungus Trichoderma harzianum to plants

Abstract

Alternative and ecological strategies are necessary and demanded for disease management in order to reduce the use of pesticides in agriculture. Thus, the use of biological control agents such as plant growth-promoting rhizobacteria (PGPR) or several strains of the beneficial fungus Trichoderma spp. to combat plant diseases is the basis of biocontrol of plant pathogens and is a good approach to reach this healthy and environmentally adequate objective.

Trichoderma is a genus that includes cosmopolitan fungi acting as biological control agents due to their ability to antagonize other fungi, using several mechanisms of action with high biotechnological value. Due to their ubiquity and rapid substrate colonization and their antifungal properties they are commonly used in agriculture in a variety of commercial biopesticides and biofertilizers, and their enzyme systems are widely used not only in agriculture but other industries such as food, paper or textile. Species of Trichoderma are frequently applied in the biocontrol of soil-borne plant pathogens due to their ability to inhibit or kill plant pathogenic fungi through the production of antifungal antibiotics and/or hydrolytic enzymes or their capacity to compete for space or nutrients. In addition to the role of Trichoderma spp. as biocontrol agents, they also have other beneficial effects on plants including stimulation of plant defences, stimulation of root development, plant growth promotion, activation of seed germination, increase of leaf greenness, increase of photosynthetic efficiency and CO₂ uptake or amelioration of abiotic stresses. Furthermore, from a biotechnological point of view, the isolation of genes from Trichoderma spp. and their subsequent transfer to a plant genome may result in a significant improvement in plant defence and resistance to abiotic stresses and may contribute in the development of agricultural biotechnology.

During the last years, several genes from Trichoderma have been isolated and their overexpression in plants has opened new perspectives in obtaining increased resistance to adverse environmental conditions. Previous reports proved that the overexpression in plants of genes encoding proteins related with plant responses to environmental stresses can improve plant tolerance. For instance, transgenic tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants overexpressing a T. harzianum endochitinase are tolerant to several plant pathogens: Alternaria alternata, A. solani, Botrytis cinerea or Rhizoctonia solani.¹ More recent studies have proved that transgenic tobacco and cotton lines that overexpress chitinases from T. harzianum conferred broad resistance to fungal and bacterial pathogens, salinity and heavy metals and Arabidopsis plants overexpressing a T. harzianum HSP70 also enhanced plant responses to abiotic stress without a reduction of plant phenotype.⁴ Similarly, Arabidopsis plants that overexpress a Trichoderma gene, Thkel1, are more tolerant to salt and osmotic stress than wild type plants, probably due to the modulation of glucosidase activity and ABA levels.⁵ As a structural feature, this type of proteins is predicted to contain a series of β-sheets representing kelch domains typically involved in protein-protein interactions⁶ and also showed some degree of similarity with hypothetical Arabidopsis proteins involved in glucosinolate metabolism as well as with myrosinase binding proteins (MBPs), main players in glucosinolate breakdown.⁷

Due to these similarities, the constitutive expression of ThKel1 in Arabidopsis plants was analyzed in order to determine if it was able to alter the expression of TGG1, the Arabidopsis protein with the highest myrosinase activity,⁸ and recently suggested to play an important role in plant responses to abiotic stress through regulation of stomata opening.⁹ As shown in Figure 1, transcript levels of TGG1 were almost undetectable in three-week-old Col-0 seedlings, while TGG1 transcripts were detected in the three independent transgenic lines. Thus, we can hypothesize that at least one of the possible explanations of the higher tolerance of 35S:ThKel1 transgenic plants to salt and osmotic stress conditions may be through activation of TGG1.

Figure 1.

Expression of the myrosinase TGG1 gene in 3-wk-old Col-0 seedlings compared to ThKel1-overexpressing plants (M1, M2 and M3). Total RNA (10 µg/line) from wild type plants and M1 to M3 transgenic plants was isolated and hybridized with a specific TGG1 probe. Bottom, ethidium bromide-stained gel showing rRNAs.

Furthermore, transient expression of this gene in onion epidermal cells subjected to osmotic stress by the addition of mannitol, also confirmed the important role of this protein in the responses to abiotic stress, since cells used as controls plasmolyzed but cells transformed with Thkel1 remained intact (Fig. 2). In conclusion, our data confirm the high value of some Trichoderma strains as a source of genes able to facilitate the achievement of producing plants resistant to abiotic stresses without an alteration of their phenotype.

Figure 2.

(A) Transient expression of the green fluorescent protein (GFP) in MS media as control. (B) Transient expression of 35S:Thkel1:GFP in MS media. (C.1) Picture at optical microscope of onion epidermal cells bombarded with the construction with the GFP construction as control, in MS media complemented with 150 mM mannitol. (C.2) Transient expression of the GFP in MS media complemented with 150 mM mannitol as control, showing autofluorescence. (D) Transient expression of 35S:Thkel1:GFP in MS media complemented with 150 mM mannitol.

Addendum to: Hermosa R, Botella L, Keck E, Jiménez JA, Montero-Barrientos M, Arbona V, et al. The overexpression in Arabidopsis thaliana of a Trichoderma harzianum gene that modulates glucosidase activity, enhances tolerance to salt and osmotic stress. J Plant Physiol. 2011;168:1295–1302. doi: 10.1016/j.jplph.2011.