Jonathan D. Walton

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Deconstructing the Cell Wall

Any microorganism that attempts to colonize a plant must contend with the cell wall. One of the most conspicuous effects of microorganisms on plant cell walls is enzymic degradation. Our interpretation of the significance of this depends on our concept of the plant cell wall, which, however, is paradoxical. On the one hand, the wall is an inert mechanical support and barrier; on the other hand, the wall is a dynamic, metabolically active organelle (Robinson, 1991, and refs. therein). The wall is a nutritional source for microorganisms and animals, yet it contains noxious peroxidases, phenolics, and activated oxygen (Cooper, 1984). The wall is mainly carbohydrate, yet its proteins are disproportionately studied (Showalter, 1993). Degradation poses another paradox. On the one hand, plant cell walls are the worlds most abundant source of Glc and other sugars; on the other hand, parts of it are resistant to degradation. Since all the polymers of a plant cell wall are eventually degraded by microorganisms, for every type of chemical bond in the wall there must be an enzyme that can cleave it. Some polysaccharidases such as a-amylase are widespread in nature; others, such as ligninase, are very restricted in their distribution.

Host-selective toxins: agents of compatibility.

20 HSTs have been documented. In general, HSTs are deter? minants of host range or specificity in that plant species, varieties, or genotypes sensitive to an HST are those that are susceptible to the producing pathogen. HSTs have been criti? cal factors in two major epidemics of crops in the United States in the 20th century, including the Southern corn leaf blight epidemic of 1970 that destroyed ~15% of that years crop. They are also important factors in several other economically sig? nificant diseases throughout the world. Studies of diseases involving HSTs led to the first elucidation of the molecular ba? sis of disease susceptibility in any interaction (Southern corn leaf blight; Dewey et al., 1988) and to the first cloning and func? tional characterization of a Mendelian disease resistance gene (Northern corn leaf spot; Johal and Briggs, 1992). Some HSTs have highly unusual chemical structures and unusual biolog? ical activities. Although some HSTs are extremely toxic, the reaction to them is often controlled by single plant genes. The study of HSTs and the diseases in which they occur continues to contribute fundamental knowledge about the processes and regulation of disease susceptibility and resistance, about ba? sic plant biochemistry through their use as specific metabolic inhibitors, about the structure and organization of secondary metabolite pathways, and about the organization of fungal ge? nomes and the evolution of new pathogen races. Because cell death is a symptom of many plant diseases, it was hypothesized early on that phytotoxic compounds con? tribute to the virulence or pathogenicity of plant pathogens. Although it is now well established that many plant pathogenic bacteria and fungi produce phytotoxic compounds, the majority of these compounds are nonselective. That is, these com? pounds affect a broader range of organisms than the producing organism infects. Some nonselective toxins, such as fusicoc? cin, trichothecene, coronatine, phaseolotoxin, syringomycin, and tabtoxin, contribute to virulence or symptom development in the diseases in which they occur (Stoessl, 1981; Ballio and

Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white rot/ brown rot paradigm for wood decay fungi

Significance Wood decay fungi have historically been characterized as either white rot, which degrade all components of plant cell walls, including lignin, or brown rot, which leave lignin largely intact. Genomic analyses have shown that white-rot species possess multiple lignin-degrading peroxidases (PODs) and expanded suites of enzymes attacking crystalline cellulose. To test the adequacy of the white/brown-rot categories, we analyzed 33 fungal genomes. Some species lack PODs, and thus resemble brown-rot fungi, but possess the cellulose-degrading apparatus typical of white-rot fungi. Moreover, they appear to degrade lignin, based on decay analyses on wood wafers. Our results indicate that the prevailing paradigm of white rot vs. brown rot does not capture the diversity of fungal wood decay mechanisms. Basidiomycota (basidiomycetes) make up 32% of the described fungi and include most wood-decaying species, as well as pathogens and mutualistic symbionts. Wood-decaying basidiomycetes have typically been classified as either white rot or brown rot, based on the ability (in white rot only) to degrade lignin along with cellulose and hemicellulose. Prior genomic comparisons suggested that the two decay modes can be distinguished based on the presence or absence of ligninolytic class II peroxidases (PODs), as well as the abundance of enzymes acting directly on crystalline cellulose (reduced in brown rot). To assess the generality of the white-rot/brown-rot classification paradigm, we compared the genomes of 33 basidiomycetes, including four newly sequenced wood decayers, and performed phylogenetically informed principal-components analysis (PCA) of a broad range of gene families encoding plant biomass-degrading enzymes. The newly sequenced Botryobasidium botryosum and Jaapia argillacea genomes lack PODs but possess diverse enzymes acting on crystalline cellulose, and they group close to the model white-rot species Phanerochaete chrysosporium in the PCA. Furthermore, laboratory assays showed that both B. botryosum and J. argillacea can degrade all polymeric components of woody plant cell walls, a characteristic of white rot. We also found expansions in reducing polyketide synthase genes specific to the brown-rot fungi. Our results suggest a continuum rather than a dichotomy between the white-rot and brown-rot modes of wood decay. A more nuanced categorization of rot types is needed, based on an improved understanding of the genomics and biochemistry of wood decay.

A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum

Race 1 isolates of Cochliobolus carbonum are pathogenic on certain maize lines due to production of a host-selective cyclic tetrapeptide, HC-toxin. Flanking HTS1, which encodes the central enzyme in HC-toxin biosynthesis, a gene was identified and named TOXA. Like HTS1, TOXA occurred only in isolates of the fungus that make HC-toxin and was present as two linked copies in most toxin-producing isolates. HTS1 and TOXA were transcribed in the opposite orientation and their transcriptional start sites were 386 bp apart. The predicted product of TOXA was a 58 kDa hydrophobic protein with 10-13 membrane-spanning regions. The sequence was highly similar to several members of the major facilitator superfamily that confer resistance to tetracycline, methylenomycin, and other antibiotics. Although it was possible to mutate one copy or the other of TOXA by targeted gene disruption, numerous attempts to disrupt both copies in a single strain were unsuccessful, suggesting that TOXA is an essential gene in strains that synthesize HC-toxin. On the basis of its presence only in HC-toxin-producing strains, its proximity to HTS1 and its predicted amino acid sequence, we propose that TOXA encodes an HC-toxin efflux pump which contributes to self-protection against HC-toxin and/or the secretion of HC-toxin into the extracellular milieu.

Endopolygalacturonase Is Not Required for Pathogenicity of Cochliobolus carbonum on Maize

A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.

Improving Enzymes for Biomass Conversion: A Basic Research Perspective

The cost of enzymes for converting plant biomass materials to fermentable sugars is a major impediment to the development of a practical lignocellulosic ethanol industry. Research on enzyme optimization with the goal of reducing the cost of converting biomass materials such as corn stover into glucose, xylose, and other sugars is being actively pursued in private industry, academia, and government laboratories. Under the auspices of the Department of Energy Great Lakes Bioenergy Research Center, we are taking several approaches to address this problem, including “bioprospecting” for superior key enzymes, protein engineering, and high-level expression in plants. A particular focus is the development of synthetic enzyme mixtures, in order to learn which of the hundreds of known enzymes are important and in what ratios. A core set comprises cellobiohydrolase, endoglucanase, β-glucosidase, endoxylanase, and β-glucosidase. Accessory enzymes include esterases, proteases, nonhydrolytic proteins, and glycosyl hydrolases that cleave the less frequent chemical linkages found in plant cell walls.

The Cochliobolus carbonum SNF1 Gene Is Required for Cell Wall–Degrading Enzyme Expression and Virulence on Maize

The production of cell wall–degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression. In Saccharomyces cerevisiae, the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF1, ccSNF1, was isolated from the maize pathogen Cochliobolus carbonum, and ccsnf1 mutants of HC toxin–producing (Tox2+) and HC toxin–nonproducing (Tox2–) strains were created by targeted gene replacement. Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected, depending on the sugar. Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. Production of HC toxin was normal in the Tox2+ ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted β-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2+ ccsnf1 mutant were reduced by 53, 24, and 65%, respectively. mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1, XYL2, XYL3, XYL4, XYP1, ARF1, MLG1, EXG1, PGN1, and PGX1. The Tox2+ ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged. The Tox2– ccsnf1 mutant also formed fewer non-spreading lesions, which also retained their normal morphology. The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C. carbonum and suggest that penetration is the single most important step at which ccSNF1 is required. The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced.

Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum.

HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 microM. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression.