Anne Osbourn

Preformed Antimicrobial Compounds and Plant Defense against Fungal Attack.

Plants produce a diverse array of secondary metabolites, many of which have antifungal activity. Some of these compounds are constitutive, existing in healthy plants in their biologically active forms. Others, such as cyanogenic glycosides and glucosinolates, occur as inactive precursors and are activated in response to tissue damage or pathogen attack. This activation often involves plant enzymes, which are released as a result of breakdown in cell integrity. Compounds belonging to the latter category are still regarded as constitutive because they are immediately derived from preexisting constituents (Mansfield, 1983). VanEtten et al. (i994) have proposed the term “phytoanticipin” to distinguish these preformed antimicrobial compounds from phytoalexins, which are synthesized from remote precursors in response to pathogen attack, probably as a result of de novo synthesis of enzymes. In recent years, studies of plant disease resistance mechanisms have tended to focus on phytoalexin biosynthesis and other active responses triggered after pathogen attack (Hammond-Kosack and Jones, 1996, in this issue). In contrast, preformed inhibitory compounds have received relatively little attention, despite the fact that these plant antibiotics are likely to represent one of the first chemical barriers to potential pathogens. The distribution of preformed inhibitors within plants is often tissue specific (e.g., Price et ai., 1987; Poulton, 1988; Davis, 1991; Fenwick et ai., 1992; Bennett and Wallsgrove, 1994), and r there is a tendency for these compounds to be concentrated in the outer cell layers of plant organs, suggesting that they may indeed act as deterrents to pathogens and pests. Some diffusible preformed inhibitors, such as catechol and protocatechuic acid (which are found in onion scales), may influence fungal growth at the plant surface. In general, however, preformed antifungal compounds are commonly sequestered in vacuoles or organelles in healthy plants. Therefore, the concentrations that are encountered by an invading fungus will depend on the extent to which that fungus causes tissue damage. Biotrophs may avoid the release of preformed inhibitors by minimizing damage to the host, whereas necrotrophs are likely to cause substantial release of these compounds. The nature and leve1 of preformed inhibitors to which a potential pathogen is exposed will also vary, depending on factors such as host genotype, age, and environmental conditions (Price et ai., 1987; Davis, 1991). There have been numerous attempts to associate natural variation in levels of preformed inhibitors in plants with resistance to particular pathogens, but often these attempts have failed to reveal any positive correlation. However, whereas preformed inhibitors may be effective against a broad spectrum of potential pathogens, successful pathogens are likely to be able to circumvent the effects of these antibiotics either by avoiding them altogether or by tolerating or detoxifying them (Schonbeck and Schlosser, 1976; Fry and Myers, 1981; Bennett and Wallsgrove, 1994; VanEtten et al., 1995; Osbourn, 1996). The isolation of plant mutants defective in the biosynthesis of preformed inhibitors would allow a direct genetic test of the importance of these compounds in plant defense. However, in most cases this approach is technically difficult because of the problems associated with screening for lossof the compounds. In the absence of plant mutants, a complementary approach involving the study of fungal mechanisms of resistance to preformed inhibitors, and of the contribution of this resistance to fungal pathogenicity to the relevant host plants, offers another route toward investigating the importance of these inhibitors in plant defense. A large number of constitutive plant compounds have been reported to have antifungal activity. Well-known examples include phenols and phenolic glycosides, unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides, and glucosinolates (reviewed in Ingham, 1973; Schonbeck and Schlosser, 1976; Fry and Myers, 1981; Mansfield, 1983; Ku6, 1992; Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994; Osbourn, 1996). More recently, 5-alkylated resorcinols and dienes have been associated with disease resistance, in this case, resistance of subtropical fruits to infection by Collefotrichum gloeosporioides (Prusky and Keen, 1993). However, only a few classes of preformed inhibitor have been studied in detail to determine their possible roles in plant defense against fungal pathogens. This review focuses on three of these classes-saponins, cyanogenic glycosides, and glucosinolates-and summarizes our current knowledge of the role of these preformed inhibitors in determining the outcome of encounters between plants and phytopathogenic fungi.

Advances in Molecular Genetics of Plant-Microbe Interactions

Section A: Interaction of Bacterial Pathogens with Plants. Section B: Rhizobium--Plant Symbiotic Interactions. Section C: Interaction of Fungi with Plants. Section D: Isolation of Plant Disease Resistance Genes. Section E: Plant Responses to Pathogens and Resistance Mechanisms. Section F: Engineered Resistance to Plant Pathogens. Index.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

Biosynthesis of triterpenoid saponins in plants.

Many different plant species synthesise triterpenoid saponins as part of their normal programme of growth and development. Examples include plants that are exploited as sources of drugs, such as liquorice and ginseng, and also crop plants such as legumes and oats. Interest in these molecules stems from their medicinal properties, antimicrobial activity, and their likely role as determinants of plant disease resistance. Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. Considerable advances have recently been made, however, in the area of 2,3-oxidosqualene cyclisation, and a number of genes encoding the enzymes that give rise to the diverse array of plant triterpenoid skeletons have been cloned. Progress has also been made in the characterisation of saponin glucosyltransferases. This review outlines these developments, with particular emphasis on triterpenoid saponins.

Saponins and plant defence — a soap story

Saponins are plant glycosides that derive their name from their soap-like properties. They occur in a great many plant species, and have been implicated as pre-formed determinants of resistance to fungal attack. A number of fungi that succeed in breaching these antimicrobial plant defences produce saponin-detoxifying enzymes. The importance of one of these (avenacinase, produced by Gaeumannomyces graminis ) in determining host range has been demonstrated. Recently, avenacinase has been shown to be closely related to another saponin-detoxifying enzyme, tomatinase, which is produced by the tomato pathogen Septoria lycopersici , suggesting that common mechanisms for saponin detoxification may be widespread in phytopathogenic fungi.

The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi

Pathogens have evolved different strategies to overcome the various barriers that they encounter during infection of their hosts. The rice blast fungus Magnaporthe grisea causes one of the most damaging diseases of cultivated rice and has emerged as a paradigm system for investigation of foliar pathogenicity. This fungus undergoes a series of well-defined developmental steps during leaf infection, including the formation of elaborate penetration structures (appressoria). This process has been studied in great detail, and over thirty M. grisea genes that condition leaf infection have been identified. Here we show a new facet of the M. grisea life cycle: this fungus can undergo a different (and previously uncharacterized) set of programmed developmental events that are typical of root-infecting pathogens. We also show that root colonization can lead to systemic invasion and the development of classical disease symptoms on the aerial parts of the plant. Gene-for-gene type specific disease resistance that is effective against rice blast in leaves also operates in roots. These findings have significant implications for fungal development, epidemiology, plant breeding and disease control.

Host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme

Antifungal saponins occur in many plant species and may provide a preformed chemical barrier to attack by phytopathogenic fungi. Some fungal pathogens can enzymatically detoxify host plant saponins, which suggests that saponin detoxification may determine the host range of these fungi. A gene encoding a saponin detoxifying enzyme was cloned from the cereal-infecting fungus Gaeumannomyces graminis. Fungal mutants generated by targeted gene disruption were no longer able to infect the saponin-containing host oats but retained full pathogenicity to wheat (which does not contain saponins). Thus, the ability of a phytopathogenic fungus to detoxify a plant saponin can determine its host range.

Metabolic Diversification—Independent Assembly of Operon-Like Gene Clusters in Different Plants

Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering.

A saponin-detoxifying enzyme mediates suppression of plant defences.

Plant disease resistance can be conferred by constitutive features such as structural barriers or preformed antimicrobial secondary metabolites. Additional defence mechanisms are activated in response to pathogen attack and include localized cell death (the hypersensitive response). Pathogens use different strategies to counter constitutive and induced plant defences, including degradation of preformed antimicrobial compounds and the production of molecules that suppress induced plant defences. Here we present evidence for a two-component process in which a fungal pathogen subverts the preformed antimicrobial compounds of its host and uses them to interfere with induced defence responses. Antimicrobial saponins are first hydrolysed by a fungal saponin-detoxifying enzyme. The degradation product of this hydrolysis then suppresses induced defence responses by interfering with fundamental signal transduction processes leading to disease resistance.

Plant-Microbe Interactions: Chemical Diversity in Plant Defense

The chemical diversity within the plant kingdom is likely to be a consequence of niche colonization and adaptive evolution. Plant-derived natural products have important functions in defense. They also have broader ecological roles and may in addition participate in plant growth and development. Recent data suggest that some antimicrobial phytochemicals may not serve simply as chemical barriers but could also have functions in defense-related signaling processes. It is important, therefore, that we should not to be too reductionist in our thinking when endeavoring to understand the forces and mechanisms that drive chemical diversification in plants.