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Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites [1]

['José Manuel Villalobos-Escobedo', 'Plant', 'Microbial Biology Department', 'The University Of California', 'Berkeley', 'California', 'United States Of America', 'Environmental Genomics', 'Systems Biology Division', 'Lawrence Berkeley National Laboratory']

Date: 2023-10

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

The rhizosphere is composed of plant roots and associated microbes, including fungi. Interactions between roots and rhizosphere bacteria have been intensely investigated, but interactions between bacteria and fungi in the rhizosphere are much less understood. Fungi in the rhizosphere, including the root endophyte, Trichoderma atroviride, have the capacity to synthesize and secrete a wide array of complex metabolites that may have a role in shaping both plant and microbe interactions. Using libraries of mutants of rhizosphere bacteria, we show that exposure to T. atroviride exudates results in a reduction in fitness in bacterial mutants carrying lesions in genes associated with antibiotic resistance and iron transport but an increase in fitness in mutants with lesions in purine biosynthesis. These results show that interactions between bacteria and fungi can be either beneficial or inhibitory, and that interactions between fungi and bacteria can display specificity, such that different bacterial species will have different responses to the presence of fungi. This study shows that T. atroviride exudates have the potential to shape microbial communities and shows the complexity of interactions between fungi and bacteria in the rhizosphere.

Funding: The work conducted in this study was funded by the m-CAFEs Microbial Community Analysis & Functional Evaluation in Soils ([email protected]), a Science Focus Area led by Lawrence Berkeley National Laboratory and supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231 (NLG and AMD). The work conducted by the U.S. Department of Energy Joint Genome Institute ( https://ror.org/04xm1d337 ), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231 (RRM and WBK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Data from BarSeq experiments for K. michiganensis M5aI, H. seropedicae SmR1, P. simiae WCS417 and P. putida KT2440 in response to exudates from WT T. atroviride and the Δtmk3 mutant are available in Dataset 2. Data from BarSeq experiments with P. simiae and P. putida upon exposure to polymyxin B or to fusaric acid are available in Dataset 3. Data obtained from hydrophilic interaction liquid chromatography-tandem mass spectrometry analyses (HILIC-MS) on exudates from WT T. atroviride and the Δtmk3 mutant are available in Dataset 5.Scripts to generate the scatter plots for comparing the fitness values in each evaluated condition are available at https://github.com/jmvillalobos/BarSeq-Bacteria-Trichoderma#:~:text=/- ,BarSeq%2DBacteria%2DTrichoderma,-Public. All fitness data presented in this work and for the four bacteria in each condition are available at the fitness browser: https://fit.genomics.lbl.gov/cgi-bin/myFrontPage.cgi .

Copyright: © 2023 Villalobos-Escobedo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

This study uncovered several critical mechanisms that govern the interactions between T. atroviride exudates and bacteria, including competition for nutrients, especially iron, and the ability to modify membrane lipids associated with tolerance to cationic antimicrobial peptides. In addition, we unexpectedly discovered that plant-associated bacteria can utilize purines secreted by T. atroviride. This investigation also revealed that a mutation in the T. atroviride Hog1-MAP kinase gene (Δtmk3) reduced expression of biosynthetic gene clusters that produce secondary metabolites. Exposure of bacterial mutant libraries to Δtmk3 exudates revealed that several bacterial mutants in genes associated with tolerance to antibiotics did not show fitness defects. Analysis of the exudate metabolome of wild type (WT) versus the Δtmk3 mutant strains showed significant differences. These data support the hypothesis that the Tmk3 pathway plays a role in the biosynthesis of secondary metabolites, including those capable of inhibiting bacterial growth. These findings underscore the intricate nature of fungal-bacterial interactions in the rhizosphere and shed new light on the complex mechanisms that regulate these interactions.

Although bacteria and fungi interact in the rhizosphere and compete for space and nutrients [ 13 ], very little is known about the molecular mechanisms of root-associated bacterial interactions with Trichoderma metabolites. Here, we used T. atroviride, which can establish a positive relationship with plant roots [ 2 , 4 , 14 ], to investigate bacterial-fungal interactions. We evaluated large mutant bacterial pools generated by random DNA barcoding mutagenesis (RB-TnSeq) [ 15 ] to identify genes important for fitness in the presence of T. atroviride exudates. We tested three different bacterial species capable of colonizing plant roots and promoting growth, Klebsiella michiganensis M5aI [ 16 ], Herbaspirillum seropedicae SmR1 [ 17 ] and Pseudomonas simiae WCS417 [ 18 ]; both K. michiganensis M5aI and H. seropedicae SmR1 can fix nitrogen but belong to different taxonomic classes. In addition, we also investigated P. putida KT2440 [ 19 ], since this is a species routinely isolated from the soil, but is not typically associated with the rhizosphere. P. putida KT2440 also has various mechanisms of resistance to toxic chemicals, potentially making it an excellent ‘biosensor’ for fungal-bacterial interactions [ 20 , 21 ].

In addition to interacting with plant roots and other fungi, Trichoderma species also interact with many species of bacteria in the rhizosphere. For example, 47 bacterial isolates from the rhizosphere were strongly inhibited by compounds secreted by T. virens and T. harzianum [ 9 ], while exudates from ten different species of Trichoderma had a negative effect on the phytopathogenic bacteria Ralstonia solanacearum and Xanthomonas campestris [ 6 ]. Some compounds produced by Trichoderma species, such as peptaibols, which are the product of nonribosomal peptide biosynthesis, have antibiotic activity [ 10 , 11 ]. Such compounds may modify the dynamics of the rhizobiome, as recently reported with T. harzianum in the rhizosphere of black pepper [ 12 ].

Species in the fungal genus Trichoderma are ubiquitous and important members of soil communities [ 1 ]. Trichoderma species can establish competitive relationships in soil by attacking other fungi, including phytopathogenic fungi [ 2 ]. Trichoderma species can also establish symbiotic relationships with plants, which generates resistance to infections and improves water and nutrient intake [ 3 ]; Trichoderma species are often used in commercial soil/seed amendments. We previously reported that the association of Trichoderma atroviride with plant roots induces changes in the plant’s gene expression and morphology, even without direct contact with the roots [ 4 ]. These phenotypic changes are partially due to the large number of secondary metabolites that T. atroviride secretes into the environment [ 5 ]. T. atroviride can also suppress the growth of other microbes, including other fungi, by the production of secondary metabolites such as epipolythiodioxopiperazines (ETPs), peptaibols, bisvertinolone, butenolides (harzianolide), pyridones (harzianopyridone), azaphilones, koninginins, steroids, anthraquinones, lactones, trichothecenes, and others compounds that work as chemical weapons [ 6 , 7 ]. T. atroviride is also a mycoparasite and can degrade and invade fungal cell walls using hydrolytic enzymes that cause necrosis of the host fungus [ 8 ].

Results

Nitrogen-fixing bacteria are strongly affected by iron restriction Fitness assays of the four bacteria showed several genes with significant negative fitness scores (gene fitness < -1 and t < -4, where t is a statistic to measure the significance of an RB-TnSeq gene fitness score [22]) when grown in T. atroviride exudates relative to uninoculated media (Fig 3). For H. seropedicae and K. michiganensis, many of the mutants in genes that showed lower fitness values were related to the TonB-ExbB-ExbD transport system (Fig 3A, 3B and S2 Dataset). The TonB-ExbB-ExbD system is mainly involved in transporting biopolymers such as siderophores that are essential for iron uptake from the environment [23,24,25]. We also detected negative fitness in mutants in a predicted Fe2+/Pb2+ permease. In T. atroviride, the production of coprogen and ferricrocin, among other siderophores, has been reported [26]. These data suggest that H. seropedicae and K. michiganensis faced greater iron limitation when grown in the presence of T. atroviride exudates, possibly due to the sequestration of iron via fungal siderophores. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Comparison of bacterial gene log2 fitness in presence and absence of Trichoderma atroviride-spent media. At least three replicates per condition were analyzed for all strains. A) Herbaspirillum seropedicae SmR1, B) Klebsiella michiganensis M5aI, C) Pseudomonas simiae WCS417, and D) P. putida KT2440. Each point on the graphs represents the fitness value associated with a disrupted gene, with the gray points indicating no significant change in fitness (|t| > 4). The unnamed genes highlighted in green are those with negative fitness in the presence of T. atroviride exudates (Fitness < -1 in spent media), while orange points indicate positive fitness values. Shown in yellow are mutants that were phenotypically rescued in the presence of exudates compared to those growing in uninoculated media. Mutations in named individual genes with negative fitness scores in other colors are noted and their general function indicated in the box. Light orange dots represent mutants of the flagellar genes (>1 log2 fitness score). https://doi.org/10.1371/journal.pgen.1010909.g003 In P. putida, like results with H. seropedicae and K. michiganensis, but unlike P. simiae, disruption of Fe acquisition genes also produced a negative fitness effect. For example, P. putida mutants in the Ferrichrome-iron receptor and nfuA genes (PP_4755 and PP_2378) showed reduced fitness in the presence of T. atroviride exudates (Fig 3D and S2 Dataset); these genes have previously been shown to be important for acquiring iron [27].

Antibiotic resistance genes are important for the growth of P. simiae in the presence of T. atroviride exudates Mutants with disruptions in four antibiotic resistance systems in P. simiae showed reduced fitness when exposed to T. atroviride exudates (Fig 3C and S2 Dataset). The most affected mutants contained insertions in four Resistance-Nodulation-cell Division (RND)-like genes (PS417_04740, PS417_04745, PS417_17290, and PS417_17295), which function as efflux pumps in bacteria [28]. RND transporters work as a tripartite system located in the inner membrane and participate in detoxification against various antibiotics such as cationic antimicrobial peptides (CAMPs) and dianionic β-lactams [29]; insertions in genes in all three components were important for fitness of P. simiae in the presence of T. atroviride exudates. We also observed that mutants in two genes encoding ABC family transporter proteins (PS417_04380 and PS417_24530) also showed reduced fitness when disrupted. These data indicate that one of the main processes triggered in P. simiae in response to T. atroviride exudates is to use efflux pumps to remove toxic metabolites from the cell. Mutants in two Lipid-Modifying Multiple Peptide Resistance genes (mprF: PS417_22845 and virJ component: PS417_22850) and several genes of the arnACDEFT operon also showed reduced fitness when the P. simiae insertional library was exposed to T. atroviride exudates (Fig 3C). The arnACDEFT operon confers resistance to polymyxin B, an antibiotic that affects the structure of the outer membrane of Enterobacteria [30,31]. Modifying lipids in the cell wall confers resistance to polymyxin B, such as modifications of lipid A with phosphoethanolamine and 4-amino-4-deoxy-L-arabinose. The results obtained in P. simiae led us to hypothesize that bacteria in the Pseudomonas genus are mainly affected by antibiotics with polymyxin B-like structure and/or mode-of-action in exudates from T. atroviride. To test this hypothesis, we evaluated the BarSeq profile of the P. putida RB-TnSeq mutant library exposed to T. atroviride exudates (Fig 3D and S2 Dataset). In contrast to our hypothesis, we did not observe reduced fitness in mutants of the arnACDEFT operon in P. putida. Indeed, a search for orthologs of these genes in the P. putida genome showed that at least 7 of the 11 genes for resistance to polymyxin B were absent (Fig A in S1 Text). However, similar to P. simiae, in P. putida a negative fitness value was observed in mutants containing insertions in the mprF and virJ genes that are predicted to be involved in resistance to CAMPs (Fig B in S1 Text) as well as mutants in the mexF and oprN gene (PP_3427 and PP_3426), which constitute a MexEF-OprN multidrug inner membrane transporter (Fig 3D and S2 Dataset).

Exposure to polymyxin B resulted in similar fitness profiles in P. simiae as exposure to T. atroviride exudates Our data using T. atroviride spent media showed that, in P. simiae, mutants in the arnACDEFT operon showed reduced fitness (Fig 3C). The arnACDEFT operon is associated with resistance to polymyxin B and colistin in Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii [32]. However, T. atroviride does not have a biosynthetic gene cluster (BGC) annotated to produce polymyxin B. To determine if the reduced fitness in mutants of P. simiae was due to the presence of antibiotics in T. atroviride exudates that potentially act like polymyxins, we first exposed wild-type P. simiae and P. putida to polymyxin B. Consistent with the hypothesis that the P. simiae arnACDEFT operon has a role in resistance to polymyxin B, the WT strain of P. simiae can resist higher concentrations of this antibiotic as compared to P. putida (Fig C in S1 Text), which lacks a number of genes in this operon. Fitness profiling experiments using BarSeq with the RB-TnSeq libraries of P. simiae in response to polymyxin B (S3 Dataset) revealed that mutants in seventeen of the 78 genes that showed reduced fitness when P. simiae was exposed to exudates were also affected by exposure to polymyxin B (Fig 4A and 4C; 2 μg/mL), including mutants in all the genes in the arnACDEFT operon. This effect was even more striking when 3 μg/mL of polymyxin B was used (Fig D in S1 Text). In contrast, BarSeq experiments with P. putida revealed that fitness defects in mutants predicted to be involved in antibiotic resistance were not identified in response to polymyxin B exposure (Fig 4B); mutants in only four of the 40 genes affected by T. atroviride exudates were also affected by exposure to polymyxin B (Fig 4D). These results suggested that the negative impact on the fitness of P. simiae mutants could be caused by antibiotics that affect the outer membrane, such as polymyxin B. However, attempts to detect polymyxin B by mass spectrometry in T. atroviride exudates were unsuccessful, suggesting the exudates might contain other compounds with similar chemical structures and/or inhibitory modes of action. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Pseudomonas simiae and P. putida log2 fitness on polymyxin B. At least three replicates per condition were analyzed for all conditions. A) BarSeq profile of P. simiae WCS417 mutant library in response to polymyxin B (2 μg/mL). B) BarSeq profile of P. putida KT2440 mutant library in response to polymyxin B (2 μg/mL). Green dots represent genes with negative fitness in response to T. atroviride exudates in spent media (SM). C) Venn diagram of genes important for fitness in T. atroviride exudates from SM and genes important for polymyxin B resistance in P. simiae. D) Venn diagram of genes important for fitness on T. atroviride SM and genes important for polymyxin B resistance in P. putida. arnC: UDP phosphate 4-deoxy-4-formamido-L-arabinose transferase (PS417_13790), arnA: UDP-4-amino-4-deoxy-L-arabinose formyltransferase (PS417_13795), arnT: 4-amino-4-deoxy-L-arabinose transferase (PS417_13805), arnD: 4-deoxy-4-formamido-L-arabinose-phospho-UDP deformylase (PS417_13800), ugd: UDP-glucose 6-dehydrogenase (PS417_13820). https://doi.org/10.1371/journal.pgen.1010909.g004

Validation of RB-TnSeq data using individual transposon mutant strains To validate fitness data indicating that P. simiae mutants in genes encoding the RND drug efflux pump and mutants in the arn operon were sensitive to T. atroviride exudates, we assayed individual transposon insertion mutants. Insertions in selected genes were verified by Sanger sequencing to confirm the insertion site and the barcode sequence (Table C in S1 Text). We tested two independent mutants in the RND major drug efflux pump (PS417_04740 and PS417_04745) and several independent P. simiae mutants in arnA and arnT. All the insertional mutants showed high sensitivity to polymyxin B and were even more negatively affected in growth by exposure to T. atroviride exudates (Fig 5A). PPT PowerPoint slide

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TIFF original image Download: Fig 5. RB-TnSeq validation using single mutants of P. simiae and P. putida in response to T. atroviride exudates. A) Eight individual mutants were isolated from the RB-TnSeq library of P. simiae; the location of insertional mutations were confirmed by sequencing (Table C in S1 Text). Two independent mutants were tested with similar results; one is shown. The profiles correlated with the results obtained from BarSeq fitness profiles. Using two different insertional lines for the mprF and oprN genes, a growth experiment for 24 hrs was performed with 0.2X spent medium in both P. simiae (B) and P. putida (C). A one-way ANOVA and a Tukey test were performed to determine statistical differences among the different strains in the same treatment (** P < 0.01). Conditions where growth was not observed are labeled NG (no growth). https://doi.org/10.1371/journal.pgen.1010909.g005 From BarSeq fitness data, P. simiae and P. putida mutants containing insertions in mprF (multiple peptide resistance factor) showed reduced fitness in response to T. atroviride exudates (Fig 3C and 3D and Tables C and D in S1 Text). Furthermore, mutants in oprN (efflux pump) showed strong fitness defects in P. putida as compared to P. simiae. We therefore tested individual insertional mutant lines in the predicted mprF and oprN genes in P. simiae and P. putida for growth when exposed to T. atroviride exudates. The growth data showed that the mprF mutants in both P. simiae and P. putida were highly sensitive to T. atroviride exudates (Fig 5; P<0.001), indicating that genes involved in resistance to CAMPs were indeed important for fitness. In contrast, P. simiae strains containing insertions in oprN showed a slight, but not significant reduction in growth (Fig 5B), while P. putida lines containing insertions in oprN showed significantly reduced growth (P<0.01) (Fig 5C). P. simiae mutants with insertions in flagellar genes showed an increase in fitness when exposed to T. atroviride exudates in BarSeq-based fitness assays (Fig 3C). However, individual mutants containing insertions in the flgE gene, a central gene in the biosynthesis of flagella, did not show increased or reduced growth in response to T. atroviride exudates (Fig 5A). In other BarSeq experiments (https://fit.genomics.lbl.gov/), mutations that affect motility often led to a fitness increase under laboratory conditions.

T. atroviride exudate and fusaric acid fitness profiles do not correlate One of the genes in the RND system of P. simiae (PS417_04745) that when mutated showed reduced fitness upon exposure to T. atroviride exudates is predicted to be a fuaA ortholog, which is part of the fuaR-fuaABC regulon involved in fusaric acid resistance in the bacterium Stenotrophomonas maltophilia [33]. Fusaric acid is a fungal polyketide-derived secondary metabolite first characterized in species within the genus Fusarium and is both phytotoxic and a mycotoxin [34]. Fusaric acid production in T. atroviride has not been reported, although a biosynthetic gene cluster with 54% similarity to the fusaric acid cluster of Fusarium verticillioides [34] was identified in the genome (Fig E in S1 Text). We therefore evaluated the fitness of P. putida and P. simiae mutant libraries to pure fusaric acid using BarSeq analysis (Fig F in SI Text and S3 Dataset). However, unlike the polymyxin results, the bacterial response to fusaric acid showed very little correlation with fitness experiments with T. atroviride exudates (including PS417_04745), even at high concentrations. These results indicated that fusaric acid was absent from T. atroviride exudates and likely does not play a role in bacterial-fungal interactions under the conditions employed in this study.

The fitness defects of mutants involved in purine synthesis are recovered in T. atroviride exudates In addition to fitness deficits in bacterial mutants associated with exposure to T. atroviride exudates, mutants in some genes that showed fitness deficits in minimal media recovered after the addition of exudates. For example, H. seropedicae, K. michiganensis and P. simiae mutants with disruptions in purine biosynthesis genes had fitness defects in minimal media, but these growth defects were mitigated when the media was supplemented with T. atroviride exudates (Fig 7B, 7C and 7D), including glycinamide ribonucleotide transformylase (GART) gene orthologs. GART catalyzes the third step in de novo purine biosynthesis, via the transfer of a formyl group to 5’-phosphoribosylglycinamide [38]. In K. michiganensis, mutants containing insertions in multiple pur genes (purK, purM, purH, and purE) were rescued by T. atroviride exudates. These results suggest that T. atroviride excretes purines into the environment, which some soil bacteria can use.

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