The importance of chorismate mutase in the biocontrol potential of Trichoderma parareesei

Abstract

Species of Trichoderma exert direct biocontrol activity against soil-borne plant pathogens due to their ability to compete for nutrients and to inhibit or kill their targets through the production of antibiotics and/or hydrolytic enzymes. In addition to these abilities, Trichoderma spp. have beneficial effects for plants, including the stimulation of defenses and the promotion of growth. Here we study the role in biocontrol of the T. parareesei Tparo7 gene, encoding a chorismate mutase (CM), a shikimate pathway branch point leading to the production of aromatic amino acids, which are not only essential components of protein synthesis but also the precursors of a wide range of secondary metabolites. We isolated T. parareesei transformants with the Tparo7 gene silenced. Compared with the wild-type, decreased levels of Tparo7 expression in the silenced transformants were accompanied by reduced CM activity, lower growth rates on different culture media, and reduced mycoparasitic behavior against the phytopathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Botrytis cinerea in dual cultures. By contrast, higher amounts of the aromatic metabolites tyrosol, 2-phenylethanol and salicylic acid were detected in supernatants from the silenced transformants, which were able to inhibit the growth of F. oxysporum and B. cinerea. In in vitro plant assays, Tparo7-silenced transformants also showed a reduced capacity to colonize tomato roots. The effect of Tparo7-silencing on tomato plant responses was examined in greenhouse assays. The growth of plants colonized by the silenced transformants was reduced and the plants exhibited an increased susceptibility to B. cinerea in comparison with the responses observed for control plants. In addition, the plants turned yellowish and were defective in jasmonic acid- and ethylene-regulated signaling pathways which was seen by expression analysis of lipoxygenase 1 (LOX1), ethylene-insensitive protein 2 (EIN2) and pathogenesis-related protein 1 (PR-1) genes.

Keywords: 2-phenylethanol; Tparo7 gene; antifungal; salicylic acid; shikimate pathway; silencing; tyrosol.

Figure 1

Enzymes and metabolites comprising the shikimate pathway in Trichoderma. Black and gray arrows indicate the existence and absence, respectively, of genetic evidence (genes were identified in the available T. reesei genome and/or previously reported steps in yeast) for a given reaction. For simplicity, the AROM protein catalyzing the steps two to six is not shown. Abbreviations: PEP, phosphoenolpyruvic acid; E4P, erytrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; DAHPS, DAHP synthase; CS, chorismate synthase; CM, chorismate mutase; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; AAAAT, aromatic amino acid amidotransferase; ASα, antranilate synthase alpha subunit; ASβ, antranilate synthase beta subunit; PAT, prephenate aminotransferase; ADT, arogenate dehydratase; ADH, arogenate dehydrogenase; APT, antranilate phosphoribosyl transferase; PAI, phosphoribosylantranilate isomerase; IGPS, indole-3-glycerol phosphate synthase; TSα, tryptophan synthase alpha subunit; TSβ, tryptophan synthase beta subunit; ICS, isochorismate synthase; ADCS, 4-amino-4-deoxychorismate synthase; ADCL, adenylsuccinate lyase; 2-PE, 2-phenylethanol; SA, salicylic acid; PABA, p-amino-benzoic acid. Major metabolites derived from the shikimate pathway are marked in squared boxes.

Figure 2

Quantification of Tparo7 transcripts in a control transformant (Tp-TC) and three silenced transformants (Tparo7-S2, Tparo7-S3 and Tparo7-S4) by real-time PCR. Values correspond to relative measurements against the Tparo7 transcript in the wild-type T. parareesei T6 (2^−ΔΔCt = 1). The experiment was carried out with mycelia grown for 48 h on PDB and transferred to MM containing 2% glucose (A) or PDB (B) for 24 h. T. parareesei T6 actin was used as an internal reference gene. Bars represent the standard deviations of the mean of three replicates. Asterisk (^*) represents statistically significant differences (P < 0.05).

Figure 3

Dual cultures of strains T6, the silenced transformants Tparo7-S3 and Tparo7-S4, and the control transformant Tp-TC of T. parareesei and the pathogens F. oxysporum (FO) (A), R. solani (RS) (B), and B. cinerea (BC) (C) on PDA medium. Plates in the center correspond to the growth of each pathogen without Trichoderma strains. Plates were incubated at 28°C for 4 days.

Figure 4

Effect of T. parareesei supernatants in the growth of F. oxysporum (FO) (A,B) and B. cinerea (BC) (C,D). Tests were carried out without (control) or with 50 μl of filter-sterilized supernatant from 48 h-PDB cultures of strains T6, Tp-TC, Tparo7-S3 and Tparo7-S4, previously boiled for 10 min (B,D) or not (A,C). Fungal growth was determined after 28°C incubation at 24, 48, and 72 h by measuring absorbance at 595 nm using a microtiter plate reader. Values are means of six replicates. Bars corresponding to every pathogen, supernatant treatment and incubation time marked with different letters are significantly different (P < 0.05).

Figure 5

Relative expression of the PR-1 (A), LOX1 (B), and EIN2 (C) genes in 4-week-old tomato leaves from seeds coated with a conidial suspension of T. parareesei T6, Tp-TC, Tparo7-S3, and Tparo7-S4. Values correspond to relative measurements against the transcripts in tomato leaves from untreated seeds (2^−ΔΔCt = 1). Tomato actin was used as an internal reference gene. Bars represent the standard deviations of the mean of three replicates. Asterisk (^*) represents statistically significant differences (P < 0.05).