Peter S. Solomon

Emergence of a new disease as a result of interspecific virulence gene transfer

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.

Host-specific toxins: effectors of necrotrophic pathogenicity.

Host‐specific toxins (HSTs) are defined as pathogen effectors that induce toxicity and promote disease only in the host species and only in genotypes of that host expressing a specific and often dominant susceptibility gene. They are a feature of a small but well‐studied group of fungal plant pathogens. Classical HST pathogens include species of Cochliobolus, Alternaria and Pyrenophora. Recent studies have shown that Stagonospora nodorum produces at least four separate HSTs that interact with four of the many quantitative resistance loci found in the host, wheat. Rationalization of fungal phylogenetics has placed these pathogens in the Pleosporales order of the class Dothideomycetes. It is possible that all HST pathogens lie in this order. Strong evidence of the recent lateral gene transfer of the ToxA gene from S. nodorum to Pyrenophora tritici‐repentis has been obtained. Hallmarks of lateral gene transfer are present for all the studied HST genes although definitive proof is lacking. We therefore suggest that the Pleosporales pathogens may have a conserved propensity to acquire HST genes by lateral transfer.

Dothideomycete–Plant Interactions Illuminated by Genome Sequencing and EST Analysis of the Wheat Pathogen Stagonospora nodorum

Stagonospora nodorum is a major necrotrophic fungal pathogen of wheat (Triticum aestivum) and a member of the Dothideomycetes, a large fungal taxon that includes many important plant pathogens affecting all major crop plant families. Here, we report the acquisition and initial analysis of a draft genome sequence for this fungus. The assembly comprises 37,164,227 bp of nuclear DNA contained in 107 scaffolds. The circular mitochondrial genome comprises 49,761 bp encoding 46 genes, including four that are intron encoded. The nuclear genome assembly contains 26 classes of repetitive DNA, comprising 4.5% of the genome. Some of the repeats show evidence of repeat-induced point mutations consistent with a frequent sexual cycle. ESTs and gene prediction models support a minimum of 10,762 nuclear genes. Extensive orthology was found between the polyketide synthase family in S. nodorum and Cochliobolus heterostrophus, suggesting an ancient origin and conserved functions for these genes. A striking feature of the gene catalog was the large number of genes predicted to encode secreted proteins; the majority has no meaningful similarity to any other known genes. It is likely that genes for host-specific toxins, in addition to ToxA, will be found among this group. ESTs obtained from axenic mycelium grown on oleate (chosen to mimic early infection) and late-stage lesions sporulating on wheat leaves were obtained. Statistical analysis shows that transcripts encoding proteins involved in protein synthesis and in the production of extracellular proteases, cellulases, and xylanases predominate in the infection library. This suggests that the fungus is dependant on the degradation of wheat macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing pycnidiospores.

The nutrient supply of pathogenic fungi: a fertile field for study

SUMMARY Phytopathogenic fungi must feed on their hosts to propagate and cause disease. Their ability to access the rich nutrient supply offered by living plants is one of the most obvious properties that distinguish pathogens from saprophytes. Successful invasion by pathogens depends as much on their ability to utilize the available nutrient sources offered by plants as on their ability to penetrate plants and evade defensive mechanisms. Here, we review current knowledge on the nature of the nutrient supplies utilized by pathogens during infection. The available evidence is rudimentary in most cases. There is much evidence to suggest that fungal metabolism can be divided into at least two phases. The first is based on lipolysis and occurs during germination and penetration of the host. The second phase uses glycolysis and predominates during the invasion of host tissue. We also propose, mainly on theoretical grounds, that a third phase of nutrition occurs late in infection during which new spores are produced. Contrary to early assumptions, the nitrogen sources available to some pathogens appear to be abundant. The idea that nitrogen starvation is a cue that controls fungal gene expression during infection may need to be reassessed. Very little is known about the micronutrient (Fe, S, P) or vitamin supply. The knowledge gained from this research may enable the design of new antifungal strategies targeting potential weaknesses in fungal metabolism and will also impact on agronomic practices.

Characterization of the Interaction of a Novel Stagonospora nodorum Host-Selective Toxin with a Wheat Susceptibility Gene

Recent work suggests that the Stagonospora nodorum-wheat pathosystem is controlled by host-selective toxins (HSTs; SnToxA, SnTox1, and SnTox2) that interact directly or indirectly with dominant host genes (Tsn1, Snn1, and Snn2) to induce disease. Here we describe and characterize a novel HST designated SnTox3, and the corresponding wheat sensitivity/susceptibility gene identified on chromosome arm 5BS, which we designated as Snn3. SnTox3 is a proteinaceous necrosis-inducing toxin between 10 and 30 kD in size. The S. nodorum isolates Sn1501 (SnToxA−, SnTox2+, and SnTox3+), SN15 (SnToxA+, SnTox2+, and SnTox3+), and SN15KO18, a strain of SN15 with a disrupted form of SnToxA, were evaluated on a population of wheat recombinant inbred lines. A compatible Snn3-SnTox3 interaction played a significant role in the development of disease caused by isolates Sn1501 and SN15KO18, with Snn2 being epistatic to Snn3. Snn3 was not significantly associated with disease caused by SN15 presumably due to the major effects observed for Snn2 and Tsn1, which were largely additive. This work introduces a fourth HST produced by S. nodorum and builds on the notion that the S. nodorum-wheat pathosystem is largely based on multiple host-toxin interactions that follow an inverse gene-for-gene scenario.

Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts

Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.

Pathogenicity of Stagonospora nodorum requires malate synthase

A gene encoding malate synthase, a key enzyme of the glyoxylate cycle, has been cloned and characterized in the necrotrophic wheat pathogen Stagonospora nodorum. Expression studies of Mls1 showed high levels of transcript in ungerminated spores whereas malate synthase enzyme activities were low. Expression studies in planta found that Mls1 transcript levels decreased ≈ 10‐fold upon germination before slowly increasing throughout the remainder of the infection. To characterize Mls1 further, the gene was disrupted in S. nodorum by homologous recombination. In the absence of any supplied carbon source, the mls1 spores were unable to germinate and consequently the mutants were non‐pathogenic. Germination and pathogenicity could be restored by the addition of either glucose or sucrose, implying that S. nodorum is reliant upon the catabolism of lipids for infection. Furthermore, analysis of lipid bodies in the mutant strain indicated that lipid mobilization and, consequently, peroxisomal β‐oxidation of fatty acids is delayed or inhibited by the disruption of the glyoxylate cycle. This study has demonstrated for the first time in a fungal phytopathogen the requirement of malate synthase for pathogenicity, suggesting that gluconeogenesis is both dependent on the glyoxylate cycle and required for infection.

SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum–wheat interaction, as well as vital information for the general characterization of necrotroph–plant interactions.

Interactions between Wine Volatile Compounds and Grape and Wine Matrix Components Influence Aroma Compound Headspace Partitioning

A full-factorial design was used to assess the matrix effects of ethanol, glucose, glycerol, catechin, and proline on the volatile partitioning of 20 volatile compounds considered to play a role in wine aroma. Analysis of variance showed that the two-way interactions of ethanol and glucose, ethanol and glycerol, and glycerol and catechin significantly influenced headspace partitioning of volatiles. Experiments were conducted to observe the effect of varied ethanol and glucose concentrations on headspace partitioning of a constant concentration of volatiles. Analysis of variance and linear regression analysis showed that the presence of glucose increased the concentration of volatiles in the headspace, whereas increasing ethanol concentration was negatively correlated with headspace partitioning of volatiles. A subsequent study assessed the effect of diluting white and red wines with water and ethanol. It was again observed that increased ethanol concentration significantly reduced the relative abundance of volatile compounds in the sample headspace. This study investigates some of the complex matrix interactions of the major components of grape and wine that influence volatile compound headspace partitioning. The magnitude of each matrix-volatile interaction was ethanol > glucose > glycerol > catechin, whereas proline showed no apparent interaction. The results clearly identify that increasing ethanol concentrations significantly reduce the headspace concentration of volatile aroma compounds, which may contribute to explaining recent sensory research observations that indicate ethanol can suppress the fruit aroma attributes in wine.

Origins of Grape and Wine Aroma. Part 1. Chemical Components and Viticultural Impacts

Wine is an ancient beverage and has been prized throughout time for its unique and pleasing flavor. Wine flavor arises from a mixture of hundreds of chemical components interacting with our sense organs, producing a neural response that is processed in the brain and resulting in a psychophysical percept that we readily describe as “wine.” The chemical components of wine are derived from multiple sources; during fermentation grape flavor components are extracted into the wine and new compounds are formed by numerous chemical and biochemical processes. In this review we discuss the various classes of chemical compounds in grapes and wines and the chemical and biochemical processes that influence their formation and concentrations. The overall aim is to highlight the current state of knowledge in the area of grape and wine aroma chemistry.