Abstract
Trichoderma reesei (Hypocrea jecorina) is widely explored in industry and its potential for using in agriculture as a biocontrol agent against phytophatogenic fungi has just began to be investigated. We have investigated the involvement of G proteins during mycoparasitism against plant pathogens. Here we described the role of GNA1, a G-alpha protein that belongs to αi group in Cell Wall Degrading Enzymes (CWDEs) production by T. reesei during antagonism against Pythium ultimum. For that, two mutants were used: Δgna1 and gna1QL (=constitutively activated version of GNA1). The gna1QL mutant of T. reesei, like the parental TU-6, inhibited the growth of P. ultimum in plate confrontation assay and grew faster than the parental TU-6 while the Δgna1 did not grow over P. ultimum. Scanning electron microscopy showed that the gna1QL mutant promoted more morphological alterations of P. ultimum cell wall than the parental TU-6 while the Δgna1 caused no effects. The mutant Δgna1 showed less CWDEs activity than gna1QL and TU-6 during in vitro cultivations. The gna1QL mutant showed a better performance in production of CWDEs such as endochitinase, N-Acetyl-β-D-glucosaminidase (NAGase), lipase and acid phosphatase, after 72 hours of incubation. However, the parental TU-6 showed higher cellulase activity than gna1QL and Δgna1. The intracellular content of cAMP in the strains after 72 hours of incubation in the presence of P. ultimum cell wall was: gna1QL (79.85 ± 12), Δgna1 (268.65 ± 8.5) and TU-6 (109.70 ± 9.2) pmol/mg protein. RT-qPCR results showed a low level of transcripts of mycoparasitism-specific genes in Δgna1 strain. We therefore suggest that the production of some CWDEs during mycoparasitism by T. reesei against P. ultimum can be mediated by GNA1 activity or cAMP levels.
Highlights
The potential of the genus Trichoderma as biocontrol agents of plant disease was first recognized by Weindling in the early 1930s [1], which described the mycoparasitic action of T. lignorum on Rhizoctonia solani and Sclerotinia sclerotiorum and its beneficial effects in control of plant pathology
In order to understand the role of GNA1 in the antagonism of T. reesei against P. ultimum, we performed a direct dual culture confrontation tests monitoring the growth of T. reesei (TU-6, gna1QL and Δgna1) over P. ultimum during 7 days
The mutant gna1QL produced more holes than TU-6, indicating that it displays a higher efficiency of antagonism/Cell Wall Degrading Enzymes (CWDEs) production
Summary
The potential of the genus Trichoderma as biocontrol agents of plant disease was first recognized by Weindling in the early 1930s [1], which described the mycoparasitic action of T. lignorum (later renamed as T. virens) on Rhizoctonia solani and Sclerotinia sclerotiorum and its beneficial effects in control of plant pathology. Biological control by Trichoderma is known as a combination of different mechanisms, among which the most important are: competition for nutrients, production of volatile and non-volatile antibiotics, coiling around the host, and production of hydrolytic enzymes [7]. The mechanism that involves the action of hydrolytic enzymes is called mycoparasitism [2] and results in penetration of the cell wall of the host fungus and utilization of its cellular contents [7]. Using proteomic approaches, we recently identified a role for α-mannosidase and arabinofuranosidase (ABFase) in mycoparasitism [12]
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