Metagenome-assembled genomes of an acid-tolerant nitrifying bacterial community isolated from a bioreactor used in ammonium scrubbers at animal-rearing facilities.

We report 12 metagenome-assembled genomes (MAGS) of a bioreactor community of acid-tolerant nitrifying bacteria. The MAGS include autotrophs in the Nitrospira genus and heterotrophs in the Xanthomonadales, Ktedonobacterales, Cytophagales, Burkholderiales, and Hyphomicrobiales. These taxonomic and genomic data provide insights into the core community members required for nitrification at low pH.

Designing amendments to improve plant performance for mine tailings revegetation.

To provide recommendations for establishment of plants on low-pH Formosa Mine tailings, two greenhouse experiments were conducted to evaluate the use of remedial amendments to improve the survival and growth of Douglas fir (Pseudotsuga menziesii) seedlings. A preliminary experiment indicated that 1% lime (by weight) raised tailings pH, permitting seedling survival. However, high rates of biosolid application (BS; 2% by weight) added to supply nutrients were phytotoxic when added with lime. A gasified conifer biochar (BC) added to tailings at 1%, 2.5%, or 5% (by weight), along with lime and BS, caused an additional increase in pH, decreased electrical conductivity (EC), and tended to increase the survival of Douglas fir. The addition of a locally sourced microbial inoculum (LSM) did not affect survival. A subsequent experiment expanded our experimental design by testing multiple levels of amendments that included lime (0.5% and 1% by weight), three application rates (0.2%, 0.5%, and 2%) of two nutrient sources (BS or mineral fertilizer), BC (0% and 2.5%), and with or without LSM. There were many interactions among amendments. In general, Douglas fir survival was enhanced when lime and BC were added. These experiments suggest that amending with lime, a nutrient source, and BC would enhance revegetation on low-pH, metal-contaminated mine tailings.

Biosynthetic Origin of Formylaminooxyvinylglycine and Characterization of the Formyltransferase GvgI.

4-Formylaminooxyvinylglycine (FVG) is an herbicidal and antibacterial nonproteinogenic amino acid produced by several strains of the Pseudomonas fluorescens species complex. It contains a unique vinyl alkoxyamine moiety with an O-N bond, and its biosynthetic origin remains unknown. Here, we show that the gvg cluster from P. fluorescens WH6 is responsible for the biosynthesis of FVG and two additional O-N bond-containing oxyvinylglycines, guanidinooxyvinylglycine and aminooxyvinylglycine. Feeding studies in the producing bacteria indicate that these compounds originate from homoserine. We identify a formyltransferase gvgI that is required for the production of FVG and characterize the activity of this enzyme in vitro toward amino acids with a side chain amine. Sequence similarity network analysis reveals that GvgI and homologues make up a distinct group from the main classes of formyltransferases.

Absence of 4-Formylaminooxyvinylglycine Production by Pseudomonas fluorescens WH6 Results in Resource Reallocation from Secondary Metabolite Production to Rhizocompetence.

Pseudomonas fluorescens WH6 produces the non-proteinogenic amino acid 4-formylaminooxyvinylglycine (FVG), a secondary metabolite with antibacterial and pre-emergent herbicidal activities. The gvg operon necessary for FVG production encodes eight required genes: one regulatory (gvgR), two of unknown functional potential (gvgA and C), three with putative biosynthetic function (gvgF, H, and I), and two small ORFs (gvgB and G). To gain insight into the role of GvgA and C in FVG production, we compared the transcriptome of knockout (KO) mutants of gvgR, A, and C to wild type (WT) to test two hypotheses: (1) GvgA and GvgC play a regulatory role in FVG production and (2) non-gvg cluster genes are regulated by GvgA and GvgC. Our analyses show that, collectively, 687 genes, including the gvg operon, are differentially expressed in all KO strains versus WT, representing >10% of the genome. Fifty-one percent of these genes were similarly regulated in all KO strains with GvgC having the greatest number of uniquely regulated genes. Additional transcriptome data suggest cluster regulation through feedback of a cluster product. We also discovered that FVG biosynthesis is regulated by L-glu, L-asp, L-gln, and L-asn and that resources are reallocated in KO strains to increase phenotypes involved in rhizocompetence including motility, biofilm formation, and denitrification. Altogether, differential transcriptome analyses of mutants suggest that regulation of the cluster is multifaceted and the absence of FVG production or its downregulation can dramatically shift the lifestyle of WH6.

Unexpected distribution of the 4-formylaminooxyvinylglycine (FVG) biosynthetic pathway in Pseudomonas and beyond.

The biological herbicide and antibiotic 4-formylaminooxyvinylglycine (FVG) was originally isolated from several rhizosphere-associated strains of Pseudomonas fluorescens. Biosynthesis of FVG is dependent on the gvg biosynthetic gene cluster in P. fluorescens. In this investigation, we used comparative genomics to identify strains with the genetic potential to produce FVG due to presence of a gvg gene cluster. These strains primarily belong to two groups of Pseudomonas, P. fluorescens and P. syringae, however, a few strains with the gvg cluster were found outside of Pseudomonas. Mass spectrometry confirmed that all tested strains of the P. fluorescens species group produced FVG. However, P. syringae strains did not produce FVG under standard conditions. Several lines of evidence regarding the transmission of the gvg cluster including a robust phylogenetic analysis suggest that it was introduced multiple times through horizontal gene transfer within the Pseudomonas lineage as well as in select lineages of Thiomonas, Burkholderia and Pantoea. Together, these data broaden our understanding of the evolution and diversity of FVG biosynthesis. In the course of this investigation, additional gene clusters containing only a subset of the genes required to produce FVG were identified in a broad range of bacteria, including many non-pseudomonads.

Phytostabilization of acidic mine tailings with biochar, biosolids, lime, and locally-effective microbes: Do amendment mixtures influence plant growth, tailing chemistry, and microbial composition?

Abandoned mine lands present persistent environmental challenges to ecosystems and economies; reclamation an important step for overcoming these challenges. Phytostabilization is an elegant and cost-effective reclamation strategy, however, establishing plants on severely degraded soils is problematic, often requiring soil amendment additions. We evaluated whether amendment mixtures composed of lime, biochar, biosolids, and locally effective microbes (LEM) could alleviate the constraints that hinder phytostabilization success. We hypothesized that 1) plants grown in tailings amended with lime, biochar, and biosolids (LBB) would establish faster and grow larger than plants grown in tailings amended with lime only, and 2) the LEM source would influence microbial community function and structure in amended mine tailings. We conducted a greenhouse study that simulated in situ conditions to measure the influence of LBB-LEM amendment blends on plant growth, plant nutrients, metal concentrations, microbial function, and microbial community structure. Blue wildrye [Elymus glaucus Buckley ssp. Jepsonii (Burtt Davy) Gould] was grown in tailings collected from the Formosa mine site amended with various combinations of LBB-LEM. The above and below ground biomass of plants grown in mine tailings amended with LBB was 3 to 4 times larger than the biomass of plants grown in tailings amended only with lime. Although the LEM addition did not influence immediate plant growth, it did affect nutrient content and altered the rhizosphere community membership. As such, it is not yet clear if LEM-driven alterations in microbial membership will advance mine reclamation strategies by improving long-term growth.

Detection of 4-formylaminooxyvinylglycine in culture filtrates of Pseudomonas fluorescens WH6 and Pantoea ananatis BRT175 by laser ablation electrospray ionization-mass spectrometry.

The oxyvinylglycine 4-formylaminooxyvinylglycine (FVG) arrests the germination of weedy grasses and inhibits the growth of the bacterial plant pathogen Erwinia amylovora. Both biological and analytical methods have previously been used to detect the presence of FVG in crude and extracted culture filtrates of several Pseudomonas fluorescens strains. Although a combination of these techniques is adequate to detect FVG, none is amenable to high-throughput analysis. Likewise, filtrates often contain complex metabolite mixtures that prevent the detection of FVG using established chromatographic techniques. Here, we report the development of a new method that directly detects FVG in crude filtrates using laser ablation electrospray ionization-mass spectrometry (LAESI-MS). This approach overcomes limitations with our existing methodology and allows for the rapid analysis of complex crude culture filtrates. To validate the utility of the LAESI-MS method, we examined crude filtrates from Pantoea ananatis BRT175 and found that this strain also produces FVG. These findings are consistent with the antimicrobial activity of P. ananatis BRT175 and indicate that the spectrum of bacteria that produce FVG stretches beyond rhizosphere-associated Pseudomonas fluorescens.

Draft Genome Sequences of Seven 4-Formylaminooxyvinylglycine Producers Belonging to the Pseudomonas fluorescens Species Complex.

Vinylglycines are nonproteinogenic amino acids that inhibit amino acid metabolism and ethylene production. Here, we report the draft genome sequences of seven isolates of Pseudomonas that produce 4-formylaminooxyvinylglycine, a compound known to inhibit the germination of grasses and the growth of specific plant-pathogenic bacteria.

Persistence of the Host-Selective Toxin Ptr ToxB in the Apoplast.

The necrotrophic fungus Pyrenophora tritici-repentis is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by P. tritici-repentis. ToxB induces chlorosis in toxin-sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 h in planta to induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin-sensitive and -insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be reisolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently labeled ToxB indicate that the toxin remains in the apoplast in toxin-sensitive and -insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.

Necrotrophic effector epistasis in the Pyrenophora tritici-repentis-wheat interaction.

Pyrenophora tritici-repentis, the causal agent of tan spot disease of wheat, mediates disease by the production of host-selective toxins (HST). The known toxins are recognized in an 'inverse' gene-for-gene manner, where each is perceived by the product of a unique locus in the host and recognition leads to disease susceptibility. Given the importance of HSTs in disease development, we would predict that the loss of any of these major pathogenicity factors would result in reduced virulence and disease development. However, after either deletion of the gene encoding the HST ToxA or, reciprocally, heterologous expression of ToxA in a race that does not normally produce the toxin followed by inoculation of ToxA-sensitive and insensitive wheat cultivars, we demonstrate that ToxA symptom development can be epistatic to other HST-induced symptoms. ToxA epistasis on certain ToxA-sensitive wheat cultivars leads to genotype-specific increases in total leaf area affected by disease. These data indicate a complex interplay between host responses to HSTs in some genotypes and underscore the challenge of identifying additional HSTs whose activity may be masked by other toxins. Also, through mycelial staining, we acquire preliminary evidence that ToxA may provide additional benefits to fungal growth in planta in the absence of its cognate recognition partner in the host.

Solution NMR structures of Pyrenophora tritici-repentis ToxB and its inactive homolog reveal potential determinants of toxin activity.

Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a ß-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent ß-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

Comparative genomics of a plant-pathogenic fungus, Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence.

Pyrenophora tritici-repentis is a necrotrophic fungus causal to the disease tan spot of wheat, whose contribution to crop loss has increased significantly during the last few decades. Pathogenicity by this fungus is attributed to the production of host-selective toxins (HST), which are recognized by their host in a genotype-specific manner. To better understand the mechanisms that have led to the increase in disease incidence related to this pathogen, we sequenced the genomes of three P. tritici-repentis isolates. A pathogenic isolate that produces two known HSTs was used to assemble a reference nuclear genome of approximately 40 Mb composed of 11 chromosomes that encode 12,141 predicted genes. Comparison of the reference genome with those of a pathogenic isolate that produces a third HST, and a nonpathogenic isolate, showed the nonpathogen genome to be more diverged than those of the two pathogens. Examination of gene-coding regions has provided candidate pathogen-specific proteins and revealed gene families that may play a role in a necrotrophic lifestyle. Analysis of transposable elements suggests that their presence in the genome of pathogenic isolates contributes to the creation of novel genes, effector diversification, possible horizontal gene transfer events, identified copy number variation, and the first example of transduplication by DNA transposable elements in fungi. Overall, comparative analysis of these genomes provides evidence that pathogenicity in this species arose through an influx of transposable elements, which created a genetically flexible landscape that can easily respond to environmental changes.

Host-selective toxins of Pyrenophora tritici-repentis induce common responses associated with host susceptibility.

Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot of wheat, produces one or a combination of host-selective toxins (HSTs) necessary for disease development. The two most studied toxins produced by Ptr, Ptr ToxA (ToxA) and Ptr ToxB (ToxB), are proteins that cause necrotic or chlorotic symptoms respectively. Investigation of host responses induced by HSTs provides better insight into the nature of the host susceptibility. Microarray analysis of ToxA has provided evidence that it can elicit responses similar to those associated with defense. In order to evaluate whether there are consistent host responses associated with susceptibility, a similar analysis of ToxB-induced changes in the same sensitive cultivar was conducted. Comparative analysis of ToxA- and ToxB-induced transcriptional changes showed that similar groups of genes encoding WRKY transcription factors, RLKs, PRs, components of the phenylpropanoid and jasmonic acid pathways are activated. ROS accumulation and photosystem dysfunction proved to be common mechanism-of-action for these toxins. Despite similarities in defense responses, transcriptional and biochemical responses as well as symptom development occur more rapidly for ToxA compared to ToxB, which could be explained by differences in perception as well as by differences in activation of a specific process, for example, ethylene biosynthesis in ToxA treatment. Results of this study suggest that perception of HSTs will result in activation of defense responses as part of a susceptible interaction and further supports the hypothesis that necrotrophic fungi exploit defense responses in order to induce cell death.

Host-selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis-wheat interaction.

Host-selective toxins (HSTs) are effectors produced by some necrotrophic pathogenic fungi that typically confer the ability to cause disease. Often, diseases caused by pathogens that produce HSTs follow an inverse gene-for-gene model where toxin production is required for the ability to cause disease and a single locus in the host is responsible for toxin sensitivity and disease susceptibility. Pyrenophora tritici-repentis represents an ideal pathogen for studying the biological significance of such inverse gene-for-gene interactions, because it displays a complex race structure based on its production of multiple HSTs. Ptr ToxA and Ptr ToxB are two proteinaceous HSTs produced by P. tritici-repentis that are structurally unrelated and appear to evoke different host responses, yet each toxin confers the ability to cause disease. This review will summarize the current knowledge of how these two dissimilar HSTs display distinct modes of action, yet each confers pathogenicity to P. tritici-repentis.

Intracellular expression of a host-selective toxin, ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1.

*ToxA, a host-selective toxin of wheat, can be detected within ToxA-sensitive mesophyll cells, where it localizes to chloroplasts and induces necrosis. Interaction of ToxA with the chloroplast-localized protein ToxABP1 has been implicated in this process. Therefore, we hypothesized that silencing of ToxABP1 in wheat would lead to a necrotic phenotype. Also, because ToxABP1 is highly conserved in plants, internal expression of ToxA in plants that do not normally internalize ToxA should result in cell death. *Reduction of ToxABP1 expression was achieved using Barley stripe mosaic virus (BSMV)-mediated, viral-induced gene silencing. The BSMV system was modified for use as an internal expression vector for ToxA in monocots. Agrobacterium-mediated expression of ToxA in a dicot (tobacco-Nicotiana benthamiana) was also performed. *Viral-induced gene silencing of ToxABP1 partially recapitulates the phenotype of ToxA treatment and wheat plants with reduced ToxABP1 also have reduced sensitivity to ToxA. When ToxA is expressed in ToxA-insensitive wheat, barley (Hordeum vulgare) and tobacco, cell death ensues. *ToxA accumulation in any chloroplast-containing cell is likely to result in cell death. Our data indicate that the ToxA-ToxABP1 interaction alters ToxABP1 function. This interaction is a critical, although not exclusive, component of the ToxA-induced cell death cascade.

Analysis of transcriptome changes induced by Ptr ToxA in wheat provides insights into the mechanisms of plant susceptibility.

To obtain greater insight into the molecular events underlying plant disease susceptibility, we studied transcriptome changes induced by a host-selective toxin of Pyrenophora tritici-repentis, Ptr ToxA (ToxA), on its host plant, wheat. Transcriptional profiling of ToxA-treated leaves of a ToxA-sensitive wheat cultivar was performed using the GeneChip Wheat Genome Array. An improved and up-to-date annotation of the wheat microarray was generated and a new tool for array data analysis (BRAT) was developed, and both are available for public use via a web-based interface. Our data indicate that massive transcriptional reprogramming occurs due to ToxA treatment, including cellular responses typically associated with defense. In addition, this study supports previous results indicating that ToxA-induced cell death is triggered by impairment of the photosynthetic machinery and accumulation of reactive oxygen species. Based on results of this study, we propose that ToxA acts as both an elicitor and a virulence factor.

A host-selective toxin of Pyrenophora tritici-repentis, Ptr ToxA, induces photosystem changes and reactive oxygen species accumulation in sensitive wheat.

Ptr ToxA (ToxA) is a proteinaceous necrotizing host-selective toxin produced by Pyrenophora tritici-repentis, a fungal pathogen of wheat (Triticum aestivum). In this study, we have found that treatment of ToxA-sensitive wheat leaves with ToxA leads to a light-dependent accumulation of reactive oxygen species (ROS) that correlates with the onset of necrosis. Furthermore, the accumulation of ROS and necrosis could be inhibited by the antioxidant N-acetyl cysteine, providing further evidence that ROS production is required for necrosis. Microscopic evaluation of ToxA-treated whole-leaf tissue indicated that ROS accumulation occurs in the chloroplasts. Analysis of total protein extracts from ToxA-treated leaves showed a light-dependent reduction of the chloroplast protein RuBisCo. In addition, Blue native-gel electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that ToxA induces changes in photosystem I (PSI) and photosystem II (PSII) in the absence of light, and therefore, the absence of ROS. When ToxA-treated leaves were exposed to light, all proteins in both PSI and PSII were extremely reduced. We propose that ToxA induces alterations in PSI and PSII affecting photosynthetic electron transport, which subsequently leads to ROS accumulation and cell death when plants are exposed to light.

The Arg-Gly-Asp-containing, solvent-exposed loop of Ptr ToxA is required for internalization.

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 x 10(6) per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

Ptr ToxA interacts with a chloroplast-localized protein.

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteinaceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.