Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Abstract

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Fig. 1

Cladogram of the phylogenetic relationship between putative GPCRs identified in the T. reesei predicted proteome (Table 2) and those previously identified in A. nidulans, A. fumigatus and A. oryzae (Lafon et al. 2006). The tree was generated using the CLUSTALX alignment

Fig. 2

Alignment of T. atroviride Gpr1–4 with predicted fungal CRL proteins from other filamentous fungi. CLUSTALX was used to align GPCR sequences from T. atroviride (Gpr1, Gpr2, Gpr3, Gpr4) with predicted proteins from T. reesei (Tr_72605, Tr_38672, Tr_28731, Tr_123806), F. graminearum (fg01861, fg07716, fg03023, fg05239, fg09693), and the N. crassa GPR–1 (Nc_GPR1) receptor. Conserved residues are shaded in black (identical) or in grey (similar). The predicted seven transmembrane domains are marked by the bars above the sequence and are numbered (TM1–TM7)

Fig. 3

Phylogeny of class V GPCRs isolated from T. atroviride (Ta_Gpr1, Ta_Gpr2, Ta_Gpr3, Ta_Gpr4) and those identified in the T. reesei (Tr_72605, Tr_38672, Tr_28731, Tr_123806) and N. crassa (Nc_GPR-1 = NCU00786.3, Nc_GPR-2 = NCU04626.3, Nc_GPR-3 = NCU9427.3) genome sequences. The sequences were aligned and a tree was generated using CLUSTALX and neighbour-joining algorithm with 1,000 bootstraps

Fig. 4

Expression of gpr1, gpr2, gpr3, and gpr4 determined by real-time RT-PCR using tef1 as a reference gene upon cultivation of T. atroviride on PDA compared to growth on PDA augmented with 5 mM cAMP (a) and on PDA compared to growth in PDB (b). To allow comparison of mRNA levels of the four GPCR-encoding genes, mRNA levels of gpr1 on PDA were arbitrarily assigned the factor 1. The transcription ratio is presented in a logarithmic scale

Fig. 5

Real-time RT-PCR analyses of gpr1, gpr2, gpr3 and gpr4 mRNA levels upon cultivation of T. atroviride on different carbon sources using act1 as a reference gene (a) and during in vitro biocontrol using sar1 as reference gene (b). The samples from cultures with glucose, glycerol and N-acetyl-glucosamine as sole carbon sources were harvested 12 h after transfer, those from cultures with chitin as sole carbon source or from cultures grown without carbon source were harvested 24 h after transfer. For monitoring gpr expression during in vitro biocontrol, T. atroviride P1 was either grown alone, or confronted with itself, or confronted with R. solani as host fungus. To allow comparison of mRNA levels of the four GPCR-encoding genes, mRNA levels of gpr1 on glucose (a) or PDA (b) were arbitrarily assigned the factor 1. The transcription ratio is presented in a logarithmic scale

Fig. 6

(a) Real-time RT-PCR analyses of gpr1 gene expression in four selected gpr1-silenced transformants. mRNA levels of gpr1 in the parental strain were assigned the factor 1. (b) Colony morphology of the gpr1-silenced transformant gpr1sil-8 compared to the parental stain T. atroviride P1 upon growth on solid medium (PDA) in the dark after 4 days at 28°C

Fig. 7

(a) Analysis of biomass formation of the T. atroviride gpr1-silenced mutant gpr1sil-8 (filled square) in comparison to the parental strain (filled diamond) after incubation for 66 h on 95 carbon sources and water using the BIOLOG microplate assay. (b–d) Growth curves of T. atroviride parental strain (filled diamond) and the gpr1-silenced mutant (filled square) on some individual carbon sources determined after 18, 24, 42, 48, 66, 72, 96 and 168 h of incubation. The values given represent the average of three analyses