Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize

Abstract

Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma-plant interactions resulting in induction of defence responses.

Trichoderma Research in the Genome Era

“…The Trichoderma genomes have now been shown to contain a large repertoire of small cysteine-rich secreted protein-like genes (67), potentially encoding hundreds of possible elicitors. The challenge is to identify which are relevant and then test the proteins for activity (11 Figure 1) (3,91,92,96). Trichoderma secondary metabolites may be grouped into peptaibols, small NRPs (e.g., gliotoxin, siderophores), polyketides, terpenes, or pyrones.…”

Isotope-Assisted Screening for Iron-Containing Metabolites Reveals a High Degree of Diversity among Known and Unknown Siderophores Produced by Trichoderma spp

“…The siderophores of Trichoderma spp. belong to the fusigen, ferrichrome, and coprogen families (54), and their orthologous NRPS gene clusters involved in siderophore synthesis (SidD and NPS6) have been identified in T. atroviride, T. reesei, and T. virens (19,54).…”

“…In the first step, raw data were automatically screened for the natural iron isotopic pattern in order to detect (putative novel) iron chelators. Therefore, the mass spectra were investigated; to be regarded as a possible iron chelator, a principal ion containing 56 Fe had to be accompanied by the corresponding 54 Fe isotopic signal at Ϫ1.99533 m/z with a maximum relative mass deviation of Ϯ5 ppm (from the calculated mass of the 54 Fe signal) in the same spectrum. Also, the relative intensity of the 54 Fe-containing ion species had to be between 4 and 7% compared to the peak originating from the 56 Fe-containing ion.…”

“…The extracted ion chromatograms (EICs) of possible identified iron chelators ( 56 Fe-and 54 Fe-containing ion species) were extracted from raw data, and the Pearson's correlation coefficients of those EICs were calculated; as a threshold, a correlation coefficient of 0.75 had to be exceeded. To achieve peak annotation, the m/z values obtained were compared with the calculated m/z values of the protonated molecule, the ammonium and the sodium adduct of siderophores present in the in-house siderophore library (maximum relative mass deviation, Ϯ5 ppm).…”

Cultura mista, manipulação química e genética de micro-organismos: estratégias para a diversificação do metabolismo secundário

“…Em tecidos vivos, foi demonstrado que uma enzima híbrida PKS-NRPS (policetídeo sintase-peptídeo sintetase não-ribossomal) é expressa exclusivamente durante a penetração do fungo Magnaporthe grisea em folhas de arroz (Oryza sativa), e provavelmente o seu produto, embora ainda não determinado, é reconhecido por cultivares resistentes, levando a ativação da resposta de defesa (BOHNERT et al, 2004). Uma enzima PKS-NRPS também está envolvida nas interações entre Trichoderma virens e raízes de milho, resultando na indução de resposta de defesa da planta (MUKHERJEE et al, 2012).…”