Functional characterization of two putative nitrogen-sensing G-protein coupled receptors of Trichoderma atroviride

Abstract

Some fungi of the genus Trichoderma are known biocontrol agents and can be used to guard plants from disease caused by various phytopathogenic fungi such as Rhizoctonia solani, Botrytis cinerea and Sclerotinia sclerotiorum. During mycoparasitism, Trichoderma directly attacks the fungal plant pathogen right after specifically recognizing it, which results in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via G-proteins have been shown to fulfill an essential function in the regulation of pathogenicity-relevant processes. However, upstream membrane receptors involved in the recognition of host-derived signals are far from being fully identified.<br />Recent studies on the transcriptomic response of the mycoparasite Trichoderma atroviride revealed 66 genes represented by 442 expressed sequence tags (ESTs) that are specifically and significantly overexpressed during the beginning of physical contact with the two plant pathogens Botrytis cinerea and Rhizoctonia solani. Interestingly, a characteristic feature of this EST-collection was the large number of genes that code for response mechanisms as a result of facing stress from nitrogen limitation when T. atroviride is confronted with its prey.<br />Upon this observation, Seidl et.al. suggested that the receptors, which sense the nitrogen status of the medium, are modulated by components derived from the host fungus and thereby simulate a nitrogen limitation signal. Expression of the seven transmembrane helix receptor of the GPCR class IV, Stm1 (Triat1: 300620), homologue of the nitrogen sensor in Schizosaccharomyces pombe was found to be induced under mycoparasitic conditions. Additionally, expression of many genes that encode proteases and oligopeptide transporters was induced during antagonism of the host.<br />Most of these proteases belong to the subtilisin-like-serine protease group encoded by the gene prb1. Action of Prb1 proteases was demonstrated to be essential in the mycoparasitic mechanisms of T.<br />atroviride. It has been suggested that the activity of these proteases on the cell wall of the prey fungus may release oligopeptides that may activate membrane receptors on T. atroviride which sense nitrogen starvation. Expression of stm1 and its paralogue, stm2 (Triat1: 238619) was studied in the wild type upon cultivation in different conditions as well as in media containing different nitrogen sources. Both stm1 and stm2 were found overexpressed at the contact zone with the host, and when cultivated in a poor nitrogen source like nitrate. In a replacement study, stm1 was overexpressed when the wild type was incubated in liquid medium with a poor nitrogen source like nitrate and upon total nitrogen starvation for several hours. Expression of stm2, on the other hand, was found to be induced when cultivated in primary nitrogen sources like glutamine and ammonium suggesting a possible explanation for the presence of two paralogue genes of these Class IV GPCRs in the T.<br />atroviride genome. In order to gain knowledge of the role of Stm1 and Stm2 in the mycoparasitic processes in T. atroviride, deletion mutants for both genes were generated and studied. ∆Stm1 and ∆Stm2 mutants exhibited reduced growth compared to the wild type, constant sporulation in light and in darkness, formation of rather little aerial mycelium and restrained conidial germination. These mutants were as well unable to antagonize R. solani, but showed no affected production of antifungal metabolites that inhibited the germination of B. cinerea conidia.<br />Expression of mycoparasitic relevant genes like ech4 and nag1 (encoding for the endochitinase 42 and the N-acetyl-D-glucosamidase I respectively) and prb1 was studied via qPCR in the wild type and the ∆Stm1 and ∆Stm2 mutants upon confrontation with themselves and with the host. ∆Stm1 showed an enhanced nag1 expression upon confrontation of the host, whereas ∆Stm2 showed a significant enhanced expression of ech42 and nag1 in a host-independent manner, suggesting this as a stress response due to the absence Stm2. In contrast, no expression of prb1 was observed in both deletion mutants, suggesting that these receptors may be fundamental in sensing host-derived signals that then trigger the expression of prb1 as a direct response to the presence of the prey.<br />Additionally, interactions of these class IV GPCRs were investigated for interactions with the three G[alpha] proteins Tga1, Tga2 and Tga3 of T.atroviride using the Split-ubiquitin membrane based yeast Two Hybrid system (MYTH). No interaction between both Stm1-like GPCRs and any of the G[alpha] proteins of T. atroviride could be observed, which may be due to an unknown methodological flaw, or to the possibility that the signal transduction through these receptors may as well occur in a G-protein independent manner. Interestingly, Stm1 showed interaction with the G[alpha] homologues of C. neoformans Gpa1, Gpa2 and Gpa3, which are highly conserved from yeasts to humans. Along with the results acquired by Chung et.al., which affirm that Stm1 in S. pombe interacts with the Tga3 homologue Gpa2, this observation supports the uncertainty about the efficiency for the MYTH system in this specific case.<br />