Identification of the ccg6 promoter in T. atroviride
To identify genes that have high levels of expression under many different physiological conditions, we mined a RNASeq database generated from T. atroviride IMI 206040 (TaWT) grown under many different environmental conditions, such as minimal medium, rich medium, high glucose, high and low light intensity, darkness, blue and red light, mechanical injury, etc., and different developmental stages [40 41, 42, 43, 44]. We found that Taccg6, a homologue of the Neurospora crassa clock-controlled gene 6 (ccg6) displayed the highest expression level under many different physiological and developmental growth conditions. Taccg6 is located in coordinates 1,051,479 to 1,052,078 in the reverse strain of contig 24 in the genome of TaWT under the gene id TRIATDRAFT_131832 (Fig. 1a) . Alignment of the predicted CCG6 protein of TaWT with orthologue sequences from other fungal species showed high similarities among proteins from different Trichoderma species and to a lower extent to that of N. crassa, which additionally had an 80 aa N-terminal extension that is missing in the proteins encoded by genes from all other fungal species analyzed (Fig.1b). The presence of this additional fragment in the N. crassa CCG6 protein suggest that it might have a different biological function than those of Trichoderma and other fungal species. Phylogenetic analysis showed that the CCG6 proteins from Trichoderma species form a discrete clade compared to those of other filamentous fungi (Fig. 1c). CCG6 proteins of Trichoderma species were analyzed using the InterProScan  and the SignalP  servers to identify putative conserved domains and signal peptides in the proteins. The analyses suggest that CCG6 belong to the SED1/SPI1 family of membrane-bound proteins that hasma non-cytoplasmic C-terminal domain constituting most of the protein sequence. The presence of a potential signal peptide with sequence MKFTAAVALAAV(A)GVSA in the N-terminal was also predicted and found to be present in all the Trichoderma CCG6 proteins analyzed (Fig. 1b).
We denominated Pccg6 to the promoter sequence of Taccg6 (submitted to the GenBank database under accession number MK887357) of T. atroviride IMI206040 (Fig.1a). To gain insight about DNA motifs potentially present and conserved between the promoter region of Trichoderma ccg6 genes, a comparative analysis of 600 bp upstream of the transcription start site of three different ccg6 genes (TaWT, Tr, T. asperellum) using the MEME suite was carried out. Interestingly, we were able to identify common putative DNA motifs distributed along the sequence between the three promoters, some of which were disposed in specific arrangements (Fig. 2). To easily compare DNA motifs present in the promoters, we organized the motifs into four groups; group 1 (motifs 1, 2, 3), group 2 (motif 4, 5, 6), group 3 (motifs 7, 8, 9), and group 4 (motifs 11 and 10) (Fig. 2). We found groups 1, 2, and 3 present in the same order in the promoters of the ccg6 gene from T. atroviride and T. asperellum (Fig. 2). In T. reesei, we found only group 1, and apparently an inverted version of group 3 present upstream with motif 5 inserted between motifs 7 and 8. In addition, group 4, comprising motifs 10 and 11, was also present in the three promoters, however, motifs 10 and 11 were next to each other in the ccg6 promoters of T. reesei and T. asperellum ccg6, immediately upstream the transcriptional start site, whereas in T. atroviride both motifs were located apart of each other (Fig. 2). We then used these DNA motifs to search for similarity against a collection of yeast transcription factor (TF) binding site motifs. We found that some of the motifs identified in Pccg6 closely resembled the binding motif of different types of TFs including zinc-finger, basic leucine zipper, and Myb-type TFs. Some of the These TFs are Haap1, Yrm1, Mig1, Mig2, Mig3, Stb5, Ino4, Ino2, among others (Table S1).
Construction of ccg6OPT::ptxD and pkiOPT::ptxD
To determine if the ccg6 sequence promoter is functional as a constitutive promoter, we selected as growth reporter gene the coding sequence of the ptxD gene from Pseudomonas stutzeri WM88 that encodes a phosphite oxidoreductase (PTXD). PTXD converts Phi into phosphate allowing organisms that express this enzyme to use Phi as a sole P source, a trait that is not present in most eukaryotic and is present only in few bacterial strains [46, 47]. Therefore, PTXD can act as a growth reporter gene, for which enzymatic assays are also available [48, 49]. However, there is no information about the functionality of the system in filamentous fungi. To test whether a 600 bp fragment of the Taccg6 promoter, containing most of the conserved array of TF binding sites, we constructed chimeric genes fusing the Taccg6 promoter with the coding sequence of ptxD (Figure S1). To be able to compare expression level driven by the ccg6 promoter with that of a known promoter, we selected Ppki1, a widely used constitutive promoter for gene expression in Trichoderma species, which was also fused to the ptxD coding sequence (Figure S1). For both constructs a codon-optimized version of the ptxD gene for expression in TaWT (submitted to the GenBank database under accession number MN434083) and the pCB1004 vector as backbone were used .
T. atroviride is unable to metabolize Phi as sole P source
In principle, the ptxD/Phi system should work for any organism unable to naturally metabolize Phi. To test the potential use of ptxD as a growth marker in T. atroviride, we first determined whether this fungal species is naturally capable of using Phi as the sole P source. With this aim, we used the Vogel’s minimal media (VMM) devoid of phosphate and supplemented with different concentrations of phosphite (Phi) or phosphate (Pi) to study the growth of a wild-type strain of T. atroviride (TaWT). For these experiments we used the two commonly sources of T. atroviride inoculum for propagation, mycelium plugs and conidia. TaWT was inoculated in solid VMM containing 1, 2, 3, 4, and 5 mM Phi as sole P source to compare its growth with that displayed in Pi-containing media (Pi) and media lacking P (No P); standard PDA rich medium was used as control. In the case of experiments using TaWT mycelium plugs, we observed a very clear and abundant growth in PDA and Pi-containing media covering an area in the dish of 59.99 and 58.45 cm2, respectively, with no statistical difference in colony area. In media lacking P, we observed abundant growth, but slightly decreased colony area (52.88 cm2) as compared to PDA and Pi controls (Fig. 3a, Table 1). TaWT growth in media lacking Pi is probably due to the presence of high amount of Pi in the agar plug itself used in the inoculum and/or to high reserves accumulated in the fungus during the initial propagation as the standard VMM medium contains 36 mM Pi. Interestingly, when the mycelium plugs were inoculated in media containing Phi, we observed that colony development was severely inhibited when compared to the growth observed in Pi, No P, and PDA media (Fig. 3a, Table 1). Inhibition of TaWT growth was Phi concentration-dependent, as determined by the area of the colony, ranging from 12.21 cm2 in 1mM Phi to less than 3 cm2 in 5 mM Phi, including the area of the plug (~2 cm2). Although non-toxic effects have been reported for Phi, growth inhibition has also been reported in plants and microalgae probably due to a competition with Pi for transporters to entry into the cell or by inhibition of enzymatic reactions that require Pi. To test whether the presence of Pi in the agar plug or Pi accumulated in the mycelia was responsible for the observed growth in media lacking Pi or supplemented with Phi, we inoculated fresh plates with mycelium plug produced from VMM plates without a P source (such as that shown in Fig. 3a). When the Pi-depleted mycelia was transferred to 2mM Phi, the growth of TaWT was completely inhibited, but when the inoculum was produced in media containing 2.5 mM Pi a certain degree of growth was observed (Figure S2), confirming that either the agar plug or Pi accumulated in the mycelia allowed growth in the presence of Phi.
When the experiments were carried out using conidia as inoculum, we observed that TaWT grew vigorously in PDA media and displayed similar colony area as when mycelia plugs were used as inoculum (Fig. 3a, Table 1). However, in VMM containing Pi, TaWT formed colonies with a less dense mycelial mat and about 14 % less area than those observed when mycelium plugs were used as inoculum. In media lacking Pi, TaWT conidia was also able to grow but with a much less dense mycelial mat with about 27 % smaller colony area than that observed in Pi containing media (Fig. 3a, Table 1). In media containing Phi as a sole P source, no growth was detected at any concentration, and in fact growth inhibition was observed in the central part of the dish were the drop containing conidia was deposited (Fig. 3a, Table 1). Similar results were observed by germinating conidia in liquid MVV supplemented with the same Phi concentrations (Fig. 3b). These results demonstrate that T. atroviride IMI 206040 is unable to use Phi as a P source, which instead exerts an inhibitory effect on its growth.
To test whether other Trichoderma species are unable to use Phi as a P source, we tested the growth of Tr and T. virens Gv29-8 (Tv) in VMM supplemented with Phi concentrations (0.25, 0.5, 0.75, 1 and 2 mM) and including Pi, No P, and PDA media as controls. TaWT was also cultured in the same media. The three Trichoderma species displayed abundant growth in PDA media showing that the conidia inoculum was viable (Fig. 4, Table 2). When the three Trichoderma species were cultured in VMM containing Pi as P source, we observed that TaWT and Tv covered most of the Petri dish with a dense mycelial mat, whereas Tr covered a smaller are of the Petri dish with less dense growth. The observed reduced growth of Tr is probably because VMM is not the optimal growth media for this Trichoderma species (Fig. 4, Table 2). When cultured in media lacking Pi, the three Trichoderma species displayed a less dense growth than that observed under Pi media and a decreased colony area of 41, 32 and 26 % for TaWT, Tr and Tv, respectively, as compared to their growth in Pi media (Fig. 4, Table 2). When conidia were inoculated in Phi-containing media, the growth of the three Trichoderma species was completely inhibited in media containing 1 and 2 mM Phi (Fig. 4, Table 2). However, TaWT and Tv displayed some degree of visible growth in 0.25- and 0.5-mM Phi (Fig. 4, Table 2). A similar behavior was observed for the three Trichoderma species when mycelium plug was used as inoculum (Figure S3, Table 2). These results show that all Trichoderma species have Pi reserves or are able to scavenge traces of Pi presents as contaminant in the media to allow some degree of growth in media devoid of Pi and, more importantly, that Tv and Tr are also unable to metabolize Phi as P source.
Phosphite metabolism can be used as a dominant growth marker in T. atroviride
To investigate whether the ccg6 promoter can be used to express heterologous genes in T. atroviride, vectors pki1OPT and the ccg6OPT were transformed into TaWT via a previously reported protocol . The hygromycin B resistance cassette present in pCB1004, in which the hygromycin B phosphotransferase gene is under control of the Amp (bla) promoter, was used as a selectable marker for the transformation process. After protoplast transformation, at least fifty primary hygromycin resistant colonies were obtained for each construct. Fifteen of these hygromycin resistant colonies were subjected to five rounds of single spore isolation until stable lines were obtained. The transformants obtained were picked out onto PDA plates containing hygromycin (100 mg×mL-1) for routine conservation.
Initial experiments using modified VMM supplemented with 1 mM Phi as the P source showed that two randomly selected hygromycin resistant clones (one per construct), ccg6OPT-3 and pki1OPT-6, were also able to use Phi as a P source (Figure S4a). To determine whether expression of ptxD has a detrimental effect on the growth of T. atroviride, we measured the radial growth, in a time-course experiment, during 72 h in standard media for two independent transgenic clones for each construct (ccg6OPT-3, and -5, and pki1OPT-6 and -5). We found that the four transgenic strains displayed a growth rate comparable to that of the TaWT control with no statistical difference, indicating that their vegetative growth was not affected by the expression of ptxD (Figure S4b). We then selected six T. atroviride transgenic lines, three harboring the ccg6OPT construct (ccg6OPT-3, -5 and -6) and three containing the pki1OPT construct (pkiOPT-1, -5 and -4), to evaluate their ability to grow in liquid media supplemented with 1 mM Phi as P source. Media supplemented with Pi and lacking a P source were used as controls. After 8 days of incubation, we observed that the six transgenic lines grew as well as the WT control in media containing Pi as a P source, whereas none of the strains grew in media devoid of a P source (Fig. 5a). In media containing Phi as a sole P source the TaWT strain was unable to sustain growth, while the four transgenic strains showed a similar growth as the WT grown in media containing Pi as a P source (Fig. 5a). These transgenic clones were also able to grow in higher Phi concentrations (2, 4 and 5 mM; Figure S5). To investigate whether the ccg6 promoter had a similar capacity to drive the expression of ptxD as that of Ppki1, three transgenic clones containing each construct were inoculated in triplicate into flaks containing liquid minimal media supplemented with Phi as a sole P source and the dry weight of the accumulated biomass determined. We found that all the strains expressing ptxD accumulate a substantial amount of biomass (between 3.7 to 5.1 mg×mL-1 DW) in media containing Phi as a sole source of P, whereas the TaWT in the same media did not accumulate a significant amount of biomass (0.23 mg×mL-1 DW) (Fig. 5b). No statistical difference in growth was detected between the growth of the clones containing the constructs ccg6OPT-3 and pki1OPT-6. These results indicate that the ccg6 sequence is functional and acts as a constitutive promoter, as it has the ability to direct a similar level of ptxD expression as that directed by the pki1 promoter, which is reflected in the capacity of the transformants for normally growing in media containing Phi as a sole P source.
To further characterize the transgenic clones, the presence of ptxD, presence, level of ptxD transcripts, and enzymatic activity were determined in different ccg6OPT-3 and pkiOPT-6 transgenic clones. Using genomic DNA, we determined by PCR that the ptxD gene is present in all the transformants analyzed (Figure S6). To corroborate that the similar growth observed for ccg6OPT and pki1OPT strains indeed represents that the ccg6 promoter can drive the expression of ptxD at a similar level, ptxD transcript levels were determined by qRT-PCR for both types of transgenic strains. We found that the two promoters lead to the accumulation of similar levels of ptxD transcript, although the ccg6 promoter appears to drive a slightly higher level of expression (Fig. 5c). PTXD enzymatic activity determined through a fluorescence-based method for NADH detection corroborated that both ccg6OPT and pki1OPT strains had similar levels of activity (Fig. 5d).
Expression of the ptxD gene does not alter the biocontrol properties of T. atroviride
To test whether the expression of the ptxD gene under either pki1 or ccg6 promoter could cause any potential change in the biological characteristics of T. atroviride, we conducted a characterization of the transgenic strains evaluating their mycoparasitism and antagonism activities. In order to evaluate the antagonistic activity of transgenic lines against phytopathogenic fungi, we performed confrontation and antibiosis assays between three selected transformants, ccg6OPT-3, -5, and -6, and pki1OPT-2, -5, and -6, against Rhizoctonia solani AG5 (RsAG5). For the confrontation experiments, we inoculated R. solani on one side of the Petri dish and on the other the TaWT or transgenic strains. In these experiments, we observed that when RsAG5 was inoculated alone, it covered at least 80 % of the Petri dish at the end of the experiment (Figure S7, Table S2). When R. solani was inoculated in the same dish with TaWT, its growth was arrested in less than 25 % of the Petri dish total area and was overgrown by the Trichoderma strain (Figure S7). TaWT grew effectively and covered more than 75 % of the Petri dish total area (Table S2). Similar results were obtained when the four transgenic strains were confronted with RsAG5; the growth of the phytopathogenic fungi was arrested and was overgrown by the transgenic ptxD-Trichoderma strains which covered almost two third of the Petri dish total area with no statistical difference between the TaWT and the transgenics (Figure S7, Table S2). Thus, the three transgenic T. atroviride strains displayed equivalent antagonism toward the pathogenic fungus to that observed for the TaWT strain.
For the antibiosis assays, TaWT was inoculated on a cellophane membrane that was placed onto the agar media. After 48 h of TaWT growth on the Petri dish, the cellophane membrane was removed to eliminate fungal mycelia and the clean dish was inoculated with RsAG5 to evaluate the effect of the metabolites released by Trichoderma into the media. In control dishes in which a clean cellophane membrane was placed, RsAG5 growth was clearly visible covering over 50% of the Petri dish surface at the end of the experiment (Figure S8, Table S3). In Petri dishes in which the cellophane membrane had TaWT, the growth of RsAG5 was completely inhibited. When the three T. atroviride transgenic strains were tested in this antibiosis assay, we found that all the strains also completely inhibited the growth of RsAG5 (Figure S8, Table S3). These results suggest that neither the use of the promoter ccg6 or the expression of the ptxD gene interfere with the biological properties of T. atroviride.
Cultivation of Trichoderma in Phi-containing media prevents the growth of contaminant bacteria
Contaminant organisms are a major barrier for the establishment of effective bioprocess, as they compete for nutrients and general resources with the organisms of interest and, thus, compromise yield of biomass and yield and quality of the bioproduct. Biological contamination is a serious constrain for the industrial use of yeast strains (i.e. S. cerevisiae, Yarrowia lipolytica) for biofuels and other fermentative processes, and the ptxD/Phi system was proven as an effective strategy for the restriction of contaminations [50, 51, 52]. To explore whether Trichoderma transformants have the capacity to outcompete contaminating organisms when grown in a Phi-supplemented medium, we designed experiments to simulate Trichoderma growth in competition with a bacterial contaminant. With this aim, one of the transgenic T. atroviride strains (ccg6OPT-3) and E. coli, as the competitor contaminant, were selected for experimentation. We established co-culture experiments by growing Trichoderma ccg6OPT-3 and E. coli together in VMM supplemented with 4 mM Phi as the sole P source, and No P and Pi as control treatments (Fig. 6a). E. coli and T. atroviride ccg6OPT-3 monocultures grown under the same conditions were also established. For E. coli, growth was determined as colony-forming units (CFU) in LB media, whereas Trichoderma biomass was measured as mycelia dry weight. Mycelium and E. coli cells were separated by filtering in Whatman filter paper.
Under control conditions without a P source, both E. coli (less than 10 x 106 CFU×mL-1) and ccg6OPT-3 (less 0.025 g DW) showed limited growth above the original inoculum in both monoculture and co-culture conditions (Fig. 6a, b). Under Pi conditions, ccg6OPT-3 showed biomass production of 0.1263 g DW growing as monoculture and 0.1 g DW when grown in co-culture with E. coli (Fig. 6b). E. coli achieved a growth of 53 x106 CFU×mL-1 growing as monoculture and 46 x 106 CFU growing as co-culture (Fig. 6c). These results indicate that E. coli and the transgenic T. atroviride have reduced growth when grown in mixed cultures probably resulting from competition for essential nutrients or the release of metabolites that inhibit the growth of each other. When Phi was supplemented as the sole P source, the growth of E. coli in monoculture was 5 to 6 times lower than that observed in media containing Pi as a P source and similar to that obtained in media lacking a P source. In the Ta/Ec mixed culture in media containing Phi as sole P source, E. coli CFUs were much lower than those observed when the bacterium was grown alone in media lacking a P source or media containing Phi as P source, indicating that the presence of Phi and an actively growing T. atroviride strain have a synergistic negative effect on the growth of E. coli. Interestingly, the biomass accumulation of ccg6OPT-3 under Phi conditions was higher, both as monoculture and in competition with E. coli, than that obtained in media containing Pi as a P source. These data show that the engineered strains of Trichoderma expressing the ptxD gene are able to outcompete the contaminant organism when grown in media containing Phi, and that the system is effective controlling bacterial contaminations.