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Fungal phytotoxins as mediators of virulence
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Fungal phytotoxins as mediators of virulence Nadine Mo¨bius1,2 and Christian Hertweck1,2 Many phytopathogenic fungi exert their destructive effects by producing and secreting toxic low molecular weight compounds. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. This review highlights effective phytotoxin-mediated strategies to distress, weaken or kill the plant host. Addresses 1 Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany 2 Friedrich Schiller University, Jena, Germany Corresponding author: Hertweck, Christian ([email protected])
Current Opinion in Plant Biology 2009, 12:390–398 This review comes from a themed issue on Biotic Interactions Edited by Xinnian Dong and Regine Kahmann Available online 14th July 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.06.004
Introduction Among the causal agents of infectious diseases of crop plants phytopathogenic fungi play an important role. Not only by causing devastating epidemics, but also through the less spectacular although persistent and significant annual crop yield losses fungal plant pathogens have a serious economic impact. Many protein factors are involved in the process of infection and the establishment of a parasitic fungal–plant interaction, such as cell wall degrading enzymes (cutinases, hydrolytic enzymes, etc.) [1]. Even so, low molecular weight phytotoxins often play a key role in infection and virulence. Typically, such fungal secondary metabolites alone reproduce some or even all of the symptoms of the disease caused by the fungal producer organisms [2]. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. Apart from improved analytical methods for the elucidation of metabolite structures, research in this field has been propelled by the availability of full genome sequences of fungal pathogens [3,4] and the application of ‘omics’ tools and bioinformatics. ‘Genome mining’ approaches aid in predicting biosynthetic pathways of yet unknown toxic metabolites in silico [5]. Molecular tools help to very effectively generate knock out of implicated genes and allow producing toxin deficient mutants for functional Current Opinion in Plant Biology 2009, 12:390–398
analyses. By studies at the biochemical level important insights into the destructive modes of action of the phytotoxins have been gained. The strategies for exerting virulence can be manifold: In general, necrotrophic pathogens use toxins to elicit plant cell death and derive nutrition from the dead tissue, whereas biotrophic pathogens rely on living plant tissue. Phytotoxins may interact with a range of cellular targets, alter gene expression or undermine membrane integrity. A number of phytotoxins inhibit the activity of plant enzymes, thereby disrupting the biosynthesis of crucial metabolites. Other fungi interfere with the plants’ physiology by producing plant hormones, such as gibberelin or gibberellic acid (GA, from the rice pathogen Gibberella fujikuroi) [6] or the auxin indole-3-acetic acid (IAA, e.g. from Ustilago maydis, Trichoderma and Moniliophthora perniciosa) [7]. Finally, plant cells may be damaged by the production of reactive oxygen species (ROS) (Figure 1). Some phytotoxins are host-specific on the genus or even on the species level and determine the host range of the fungus by targeting specific enzymes or metabolic pathways [8]. Such host-specific toxins (HSTs) induce pathogenicity only in the host species, where a gene product is the direct or indirect target of the toxin. Nearly all fungi producing HST (i.e. fungi of the genera Alternaria, Cochliobolus, Leptosphaeria, Venturia, Ascochyta and Pyrenophora) belong to the order Pleosporales and appear to have a tendency to lateral gene transfer [1,2]. Finally, it should be noted that already in the primary infection process secondary metabolites can play an important role. Many fungal pathogens penetrate plant leaves from a specialized cell, the appressorium. The appressorium of Magnaporthe oryzae, a rice blast pathogen, is melanized by oxidative polymerization of polyketide precursors and forms an effective barrier to solute movement. Water is entering and an enormous turgor pressure of approximately 80 atmospheres is produced. The resulting force is usually applied to gain entry into plant tissue [9].
Shedding light on plant-damaging photosensitizers Various phytopathogenic fungi produce so-called photosensitizers to generate ROS and thus impair plant cells by induction of apoptosis and damage to membrane lipids. This mechanism has been observed for metabolites sharing a 3,10-dihydroxy-4,9-perylenequinone chromophore, such as cercosporin and elsinochrome (Figure 2). After absorption of light energy, perylenequinones adopt an activated triplet state. The radical can then react with www.sciencedirect.com
Fungal phytotoxins as mediators of virulence Mo¨bius and Hertweck 391
Figure 1
Overview on the cellular targets and the mode of action of several fungal phytotoxins, GDC, glycine decarboxylase; CerS, ceramide synthase, ER, enoyl reductase; HDAC, histone deacetylase complex; NO, nitric oxide; PCD, programmed cell death; PM, plasma membrane.
oxygen to form ROS (O2 , H2O2) and singlet oxygen or induce oxidative lipid decomposition, thus causing major damages to the host cell membrane [10]. Cercosporin is produced by Cercospora species that infect corn, soybean, coffee and other plants. The light-induced damage leads to leakage of nutrients in the intracellular space, thus making them available for fungal hyphae. The fungus protects itself probably via toxin export and quenchers [10]. A polyketide synthase, CTB1, plays a key role in cercosporin biosynthesis in Cercospora nictonianae, as CTB1 mutants cause fewer necrotic lesions on tobacco leaves [11]. Interestingly, expression of the CTB1 gene is highly regulated by light. Disruption of ctb4, a gene coding for a putative membrane transporter, results in decreased cercosporin emission and reduced virulence against tobacco cells [11]. The red-pigmented elsinochromes are produced by Elsinoe fawcettii when exposed to light [12]. Citrus cells, when in contact with the fungus, are rapidly killed whereas www.sciencedirect.com
addition of b-carotene or superoxide dismutase dampens plant cells damage. The polyketide synthase gene Efpks1 essential for elsinochrome production has been identified by gene disruption, and the resulting elsinochrome-negative mutant has a significantly reduced ability to form lesions in lemon leaves [12]. Another light-dependent plant-damaging mechanism has been identified in the context of Ramularia collocygni leaf spot disease on barley. Rubellin D (Figure 2), an anthraquinone derivative, induces peroxidation of alphalinoleic acid in a light dependent manner, predominantly caused by singlet oxygen formation [13]. Furthermore, chlorophyll bleaching was observed in toxin-treated leaves. Interestingly, ferrous ions enhance the rubellin-induced reaction, but repress the action of cercosporin [13].
Protein targeting Epipolythiodioxopiperazines (ETPs) contain a characteristic internal di-sulphide or tri-sulphide bridge (Figure 2). Current Opinion in Plant Biology 2009, 12:390–398
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Figure 2
Structures of apoptosis-inducing phytotoxins victorin and DON, the protein-attacking mycotoxins gliotoxin and sirodesmin (ETP), the actin skeleton targeting cytochalasin B and the photosensitizers cercosporin, elsinochrome A and rubbellin D.
Although the exact mode of action is not yet fully understood, an involvement of this structural feature in protein conjugation [14] or generation of ROS via redox cycling is plausible [14]. Prominent examples of such toxins are sirodesmin PL produced by Leptosphaeria maculans and gliotoxin produced by Trichoderma spp. and Aspergillus fumigatus [15]. A mutant with a disruption of the bimodular NRPS gene (sirP) of L. maculans stalls the production of sirodesmin PL, causes fewer lesions and is half as effective as the wild-type in colonizing stems of Brassica napus (Canola) [15].
Phytotoxins affecting membrane integrity Toxin-mediated inhibition of enzymes involved in lipid biosynthesis is a common strategy applied by phytopathogenic fungi. Important examples are the closely related toxins fumonisin (from Fusarium spp.) and AALtoxin (from Alternaria alternata), which act as sphingosine analogs and inhibit sphinganine–N-acetyltransferase and ceramide synthase (Figure 3) [16,17]. In this way, lipid biosynthesis is hampered, resulting in a perturbed membrane ordering and increased membrane permeability. It Current Opinion in Plant Biology 2009, 12:390–398
should also be noted that toxin-induced changes in ceramide metabolism may have severe consequences for a variety of regulatory processes, as ceramides are involved in intracellular signaling pathways. Gibberella moniliformis, the cause of maize seedling blight, produces fumonisin B1 as one of its virulence factors. Although fumonisin-insensitive maize strains are not resistant to infection, systemic colonization of seedlings is reduced [18]. Cyperin (Figure 3), a diphenyl ether phytotoxin produced by several fungal plant pathogens, interferes with lipid biosynthesis by inhibiting enoyl reductase (ER) [19]. Cyperin is bound to the ER active site by p-p stacking of a phenyl ring and the nicotinamide ring of NAD+. At high concentrations cyperin also blocks protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis [19]. A straightforward way of membrane damage is exerted by the fungus Cercospora beticola using so-called beticolins (Figure 3) or yellow toxins. The polyketides can selfassemble into multimeric structures and form ion www.sciencedirect.com
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Figure 3
Molecules targeting membrane integrity. Sphingosin and phytotoxic analogs AAL-toxin and fumonisin; structure of cyperin, an inhibitor of enoyl reductase and of the pore-forming toxin beticolin 0.
channels in the host membrane (Figure 1). Effects of the pore formation are dramatic loss of solutes, inhibition of ATP-dependent H+-transport and membrane depolarization in various plant species [20].
Taking what’s not given—siderophores in virulence Upon infection – and in particular after penetration – a fungus becomes dependent on the availability of www.sciencedirect.com
nutrients inside of the host. Iron is an essential nutrient and the major redox mediator in cellular processes and thus essential for survival of the pathogen [21]. Iron acquisition is carried out via the secretion and subsequent uptake of low-molecular-weight chelators, so called siderophores, like ferricrocin (A. brassicicola) or triacetylfusarinine C (TAFC, F. gramineum, Figure 4). Mechanisms for iron uptake can be regarded as virulence determinants that lead to iron depletion in the host. Deletion of genes Current Opinion in Plant Biology 2009, 12:390–398
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Figure 4
Structures of molecules causing energy breakdown in plant cells, and the siderophore triacetylfusarinine C.
coding for non-ribosomal peptide synthetases involved in siderophore biosynthesis, for example nps6 (C. heterostrophus) and sid1 (F. gramineum), led to a reduction in virulence to the host plant and to a simultaneous increase in ROS sensitivity in C. heterostrophus. The defects could be compensated by exogenous application of iron [21]. By contrast, the biotroph U. maydis relies on reductive iron uptake rather than on siderophores. Deletion of fer1 and fer2 (encoding a high-affinity iron permease and an iron multicopper oxidase) proved to be much less virulent [22].
calcium has been observed [24]. The toxin is assembled by action of two polyketide synthases (PKSs). One PKS provides the polyketide starter unit for the second PKS, which produces the mature T-toxin molecule. A third biosynthetic gene codes for a decarboxylase (DEC1) [25]. These genes reside in AT-rich DNA that is unique to T-toxin-producing strains (race T). The biosynthetic genes have probably undergone lateral gene transfer, as race T harbors an additional 1.2 Mb of DNA compared with the weakly pathogenic race 0 lacking the Tox1 locus [24].
Energy breakdown
An energy breakdown may also be caused by phytotoxins that affect the integrity of plant plasma membranes (see above), which results in an increased membrane permeability and nutrient leakage, or target H+-ATPase, thus disrupting the electrochemical gradient. It has been shown that fusicoccin, a glycosylated diterpene, augments potassium uptake with concomitant proton extrusion in rice plants, which leads to an increased extracellular acidification [26].
The blockage of ATP-hydrolysis leads to complete energy breakdown in the plant cell. Tentoxin (Figure 4), a host-selective toxin, produced by Alternaria species targets this energy transfer process in the chloroplast. The cyclic tetrapeptide blocks ATP hydrolysis by binding to the surface between the a and b subunits of chloroplast ATPase. The crystal structure of spinach chloroplast F1 with bound tentoxin was solved, and it was shown that a single molecule of the toxin affects ADP release in a non-competitive mechanism [23]. T-toxin, a polyketide synthesized by Cochliobolus heterostrophus, is associated with high virulence on certain genotypes of maize. It selectively targets T cytoplasm mitochondria mediated by the T-urf13 protein, which results in conformational changes and pore formation followed by mitochondrial swelling. Changes in oxidative phosphorylation and respiration as well as leakage of nutrients and Current Opinion in Plant Biology 2009, 12:390–398
Binding of fusicoccin to the plant plasma membrane H+ATPase is mediated by a regulatory protein belonging to the 14-3-3 family. A stable complex with the C-terminus of the H+-ATPase is formed, leading to its permanent activation and irreversible stomata opening. Its mode of action has been elucidated by X-ray crystallographic analysis of the ternary complex of fusicoccin, a plant 14-3-3 protein, and a phosphopeptide derived from the www.sciencedirect.com
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C-terminus of H+-ATPase. Structural analysis and isothermal titration calorimetry indicated that peptide and toxin mutually increase each other’s binding affinity through filling a cavity in the interaction surface [27]. In Arabidopsis thaliana cells, fusicoccin induces an H+ATPase state-independent increase in the extracellular H2O2 level. Measurement on exogenous catalase activity indicated a reduced capability of the cells to degrade H2O2 formed in cell-free media. Apparently an as yet unidentified factor accumulates in the incubation medium of cells treated with fusicoccin, which acts as a noncompetitive catalase inhibitor and is able to reduce the cell’s capacity for H2O2 scavenging [28]. Apart from the mode of action, also the fusicoccin biosynthetic machinery is quite intriguing: PaFS, a multifunctional enzyme of Phomopsis amygdali, plays a key role with prenyltransferase (responsible for condensation of isoprene units) and terpene cyclase (cyclization of C-20 precursor) activity. Both enzymatic functions required for diterpene biosynthesis are normally carried out by two independent enzymes [29].
Strategies to trigger apoptosis The induction of apoptosis, or programmed cell death (PCD) in plants, is a major strategy of phytopathogenic fungi to acquire nutrients from plants. However, the underlying mechanisms can be manifold. Trichothecenes, such as deoxnivalenol (DON), are nonvolatile sesquiterpenoids produced by Fusarium graminearum (Figure 2). DON is a host-specific virulence factor that is frequently found in contaminated cereal crops [30]. In Arabidopsis DON inhibits translation without inducing a plant defense response [30]. The first gene of the trichodiene pathway catalyzes the cyclization of farnesyl pyrophosphate to trichodiene. Disruption of this trichodiene synthase (a terpene cyclasetype enzyme) gene results in a DON-non-producing strain. Conidia of the mutant are still able to infect but disease symptoms are significantly reduced. Mutants deficient in crucial toxin biosynthetic genes exhibited reduced virulence on wheat seedlings but were unaffected with respect to causing disease on barley [31]. These results indicate that these fungal toxins confer host specificity to Fusarium pathogenicity. Notably, wheat infection by the mutants is associated with thickening of the cell wall in the rachis node, a plant induced defense [31]. Self-resistance to DON is conferred by the enzyme trichothecene 3-O-acetyltransferase (Tri101), which transforms DON into 3-acetyldeoxynivalenol (3-ADON). Heterologous expression of Tri101 in rice plants significantly reduced phytotoxic effects [32]. Victorin (Figure 2) is a cyclic pentapeptide produced by the fungus Cochliobolus victoriae that causes Victoria oat blight [33]. The toxin enters mitochondria by a mitowww.sciencedirect.com
chondrial permeability transition (MPT) and binds to Pprotein of the mitochondrial matrix, a subunit of the glycine decarboxylase complex (GDC, part the photorespiratory cycle). After incubation with victorin a rapid response is observed, in particular DNA laddering, lipid oxidation and cleavage of RUBISCO, followed by the inhibition of photorespiration [34]. Recently, victorininsensitive mutants were isolated, with resistance conferred to specifically by a mutation in liv1 (locus of insensitivity to victorin1). The gene product is thioredoxin h5 (ATTRX5), a member of a large family of disulphide oxidoreductases [34]. Sensitivity to the toxin in Arabidopsis thaliana relies on the gene lov1 that codes for a coiledcoil–nucleotide binding site leucine-rich repeat protein. Ironically, lov1 is a member of the NBS–LRR resistance gene family [35]. Finally, PCD can also be induced by the above-mentioned fusiccocin. An apoptotic-like form of PCD that involves typical apoptotic characteristics like chromatin condensation, Cytochrome c release and DNA laddering and is inhibited by cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria [36].
Breaking the cell’s bones—destroying the actin skeleton Disaggregation of the cytoskeleton can ultimately also lead to PCD and is another plant-damaging effect caused by fusiccocin (see above). Actin depolymerization seems to be mediated by induction of NO production and can be decreased by the addition of NO scavengers or actin stabilizing drugs [36]. By contrast, toxins belonging to the family of cytochalasans (gr.: kytos – cell, and chalasis – relaxation) specifically bind to actin filaments, thus blocking cytokinesis, while mitosis remains unaffected [37]. This structurally diverse group of polyketide–amino acid hybrids includes, among others, cytochalasin A, cytochalasin B (phomin), and chaetoglobosins, which are particularly active representatives (Figure 2) [37]. The molecular basis for chaetoglobosin A and C biosynthesis was elucidated in Penicillium expansum. A PKS–NRPS hybrid synthetase (CheA) assembles the linear polyketide–amino acid backbone that finally undergoes a Diels–Alder reaction to yield the tricyclic framework [38]. Interestingly, related genes coding for PKS-NRPS hybrids are present in the genome [3] of Magnaporthe oryzae. The M. oryzae ACE1 avirulence gene encodes a putative hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), with the structure of the biosynthetic product not yet elucidated. Thus, it is not clear whether the product of this gene will affect the actin cytoskeleton. Expression of ACE1 occurs early during infection and coincides with penetration of the cuticle [39]. Current Opinion in Plant Biology 2009, 12:390–398
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To sight the core-transcription factors, epigenetic modifiers and antimitotic agents Manipulation of the replication machinery and altering gene expression profiles are other important effects mediated by secreted fungal compounds. An important example is Ustilago maydis, a ubiquitous biotrophic fungus and maize pathogen. While its complete genome sequence has revealed only few secondary biosynthetic genes [4], it was found that auxin (indole-3-acetic acid, IAA) is produced. Since the concentration of auxin in U. maydis induced tumors is significantly higher (up to 20fold) in tumor tissue than in the surrounding plant tissues, fungal production of this plant growth hormone was implicated in tumor induction [40]. According to the current model IAA is involved in targeted protein degradation of the Aux/IAA transcriptional repressors via the auxin receptor TIR1 and thereby regulates transcription [41]. However, a quadruple mutant that is unable to produce IAA in culture was unaffected in tumor induction, suggesting the elevated IAA levels in tumor tissue are likely to result from fungus-induced changes in plant hormone levels [40]. As the biotrophic lifestyle requires efficient protection against plant defense reactions. Notably, in the genome of U. maydis a gene coding for a Yap1-related protein has been identified that serves as the central regulator providing S. cerevisiae. In U. maydis this transcription factor is involved in detoxification of plant produced ROS and fundamental for full virulence [42]. Another growth-regulating phytohormone is the already mentioned gibberellin (GA) that belongs to a group of diterpenoid acids responsible for stem elongation and seed germination [43]. It was first isolated from the pyhtopathogenic fungus Gibberella fujikuroi, which causes the rice plant disease Bakanae whose most prominent characteristics are strongly elongated seedlings [6]. The plant and fungal biosynthetic pathways differ only in the
last steps [6]. Recently the GA receptor GID1 has been identified in Arabidopsis. GID1 is located in the nucleus and enables degradation of a repressor bound to GAdependent transcription factors through ubiquitin ligation and leads to gene transcription [43]. Another mechanism to alter gene expression involves epigenetic modification. This strategy is employed by Cochliobolus carbonum, a fungus that is highly virulent on certain maize genotypes. C. carbonum produces HC-toxin, a cyclic tetrapeptide containing D-amino acids (Figure 5), and is highly virulent on certain genotypes of maize. Production of the host-selective compound is under the control of a complex locus (tox2) coding for HTS1 (HC-toxin synthetase), an NRPS [44]. The putative HCtoxin efflux carrier encoded by toxA is probably involved in a self-protection mechanism. The toxin is an inhibitor of histone deacetylases (HDACs) leading to hyperacetylation and thereby to changes in gene expression in the plant. In vitro kinetic studies revealed that the inhibition is uncompetitive and reversible [45]. HC-toxin stimulates the uptake of organic and inorganic molecules such as nitrate into the maize roots. Resistant maize plants carry the Hm1 resistance gene that encodes an HC-toxin reductase (HCTR) [45]. Chromosomal segregation is of vital importance for cell division and growth, and its inhibition is an effective strategy of a pathogenic organism. The antimitotic agent rhizoxin (Figure 5), a macrocyclic polyketide, is known as the virulence factor of the rice seedling blight fungus Rhizopus microsporus. Rhizoxin binds to b-tubulin, thus preventing heterodimerization with a-tubulin and consequently the formation of microtubules [46]. Only recently, it was found that the toxin is not produced by the fungus, but by bacterial endosymbionts that reside within the fungal cytosol [46]. Isolation and cultivation of the symbionts clearly demonstrated that the toxin complex is produced by the endofungal bacterium
Figure 5
Molecules targeting processes in the nucleus; phytotoxins acting as epigenetic modifier (HC-toxin) or blocking mitosis (rhizoxin). Current Opinion in Plant Biology 2009, 12:390–398
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Burkholderia rhizoxinica [47]. Furthermore, the molecular basis for rhizoxin production was elucidated by sequencing a gene cluster coding for a modular PKS–NRPS assembly line in the bacterial genome [48]. A specific mutation of b-tubulin confers resistance to the fungal host and is probably a precondition for the establishment of the symbiosis [49]. This is the first described case where a phytotoxin employed by a fungus is actually produced by endosymbionts. Here, the pathogenic relationship between the plant and the infecting fungus is extended to a tripartite system including a symbiotic alliance of fungus and bacterium. The fungus benefits from plant nutrients but is itself dependent on bacteria for toxin production and even for reproduction as spore formation can only occur in the presence of the bacterial symbionts [50].
Conclusion In conclusion, phytopathogenic fungi employ an array of strategies to distress, weaken or kill the host plant in order to gain access to nutrients. The captivating structural and mechanistic diversity of the toxins teaches us a lesson on the complexity of pathogenic relationships—up to the point where a metabolically lean fungus hosts a bacterium for toxin production. Understanding toxin biosynthesis pathways and their regulation, the modes of action and how this relates to fungal virulence will not only help to gain new insights into cellular processes in general but is also a stepping stone to develop ways to protect plants from fungal infections. With the help of advanced genomics, proteomics and analytical skills we will soon understand more about phytotoxins as mediators of plant virulence.
Acknowledgment The authors are grateful for financial support of original research in this area by the Jena School for Microbial Communication (JSMC).
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35. Lorang JM, Sweat TA, Wolpert TJ: Plant disease susceptibility conferred by a ‘resistance’ gene. Proc Natl Acad Sci 2007, 104:14861-14866. Intriguing observation that a ‘resistance gene’ confers susceptibility to a phytotoxin. 36. Malerba M, Contran N, Tonelli M, Crosti P, Cerana R: Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells. Physiol Plant 2008, 133:449-457. 37. Berestetskiy A, Dmitriev A, Mitina G, Lisker I, Andolfi A, Evidente A: Nonenolides and cytochalasins with phytotoxic activity against Cirsium arvense and Sonchus arvensis: a structure– activity relationships study. Phytochemistry 2008, 69:953-960. 38. Schuemann J, Hertweck C: Molecular basis of cytochalasan biosynthesis in fungi: gene cluster analysis and evidence for the involvement of a PKS–NRPS hybrid synthase by RNA silencing. J Am Chem Soc 2007, 129:9564-9565. 39. Fudal I, Collemare J, Bohnert HU, Melayah D, Lebrun MH: Expression of Magnaporthe grisea avirulence gene ACE1 Is connected to the initiation of appressorium-mediated penetration. Eukaryotic Cell 2007, 6:546-554. 40. Reineke G, Heinze B, Schirawski J, Buettner H, Kahmann R, Basse CW: Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation. Mol Plant Pathol 2008, 9:339-355. 41. Kepinski S, Leyser O: The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 2005, 435:446-451. 42. Molina L, Kahmann R: An Ustilago maydis gene involved in H2O2 detoxification is required for virulence. Plant Cell 2007, 19:2293-2309. 43. Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh H, Katoh E, Kobayashi M, Chow T-Y, Hsing Y-I, Kitano H, Yamaguchi I et al.: Gibberellin insensitive DWARF1 encodes a soluble receptor for gibberellin. Nature 2005, 437:693-698. 44. Walton JD: HC-toxin. Phytochemistry 2006, 67:1406-1413. 45. Baidyaroy D, Brosch G, Graessle S, Trojer P, Walton JD: Characterization of inhibitor-resistant histone deacetylase activity in plant-pathogenic fungi. Eukaryot Cell 2002, 1:538-547. 46. Partida-Martinez LP, Hertweck C: Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 2005, 437:884-888. This is the first reported case where a fungus-derived phytotoxin is in fact not produced by the fungus, but by bacterial endosymbionts residing within the fungal cytosol. 47. Scherlach K, Partida-Martinez LP, Dahse HM, Hertweck C: Antimitotic rhizoxin derivatives from a cultured bacterial endosymbiont of the rice pathogenic fungus Rhizopus microsporus. J Am Chem Soc 2006, 128:11529-11536. 48. Partida-Martinez LP, Hertweck C: A gene cluster encoding rhizoxin biosynthesis in ‘Burkholderia rhizoxina’, the bacterial endosymbiont of the fungus Rhizopus microsporus. Chembiochem 2007, 8:41-45.
33. Curtis MJ, Wolpert TJ: The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity. Plant J 2004, 38:244-259.
49. Schmitt I, Partida-Martinez LP, Winkler R, Voigt K, Einax E, Dolz F, Telle S, Woestemeyer J, Hertweck C: Evolution of host resistance in a toxin-producing bacterial-fungal alliance. Isme J 2008, 2:632-641.
34. Sweat TA, Wolpert TJ: Thioredoxin h5 is required for victorin sensitivity mediated by a CC–NBS–LRR gene in Arabidopsis. Plant Cell 2007, 19:673-687.
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Current Opinion in Plant Biology 2009, 12:390–398
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Fungal phytotoxins as mediators of virulence Nadine Mo¨bius1,2 and Christian Hertweck1,2 Many phytopathogenic fungi exert their destructive effects by producing and secreting toxic low molecular weight compounds. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. This review highlights effective phytotoxin-mediated strategies to distress, weaken or kill the plant host. Addresses 1 Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany 2 Friedrich Schiller University, Jena, Germany Corresponding author: Hertweck, Christian ([email protected])
Current Opinion in Plant Biology 2009, 12:390–398 This review comes from a themed issue on Biotic Interactions Edited by Xinnian Dong and Regine Kahmann Available online 14th July 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.06.004
Introduction Among the causal agents of infectious diseases of crop plants phytopathogenic fungi play an important role. Not only by causing devastating epidemics, but also through the less spectacular although persistent and significant annual crop yield losses fungal plant pathogens have a serious economic impact. Many protein factors are involved in the process of infection and the establishment of a parasitic fungal–plant interaction, such as cell wall degrading enzymes (cutinases, hydrolytic enzymes, etc.) [1]. Even so, low molecular weight phytotoxins often play a key role in infection and virulence. Typically, such fungal secondary metabolites alone reproduce some or even all of the symptoms of the disease caused by the fungal producer organisms [2]. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. Apart from improved analytical methods for the elucidation of metabolite structures, research in this field has been propelled by the availability of full genome sequences of fungal pathogens [3,4] and the application of ‘omics’ tools and bioinformatics. ‘Genome mining’ approaches aid in predicting biosynthetic pathways of yet unknown toxic metabolites in silico [5]. Molecular tools help to very effectively generate knock out of implicated genes and allow producing toxin deficient mutants for functional Current Opinion in Plant Biology 2009, 12:390–398
analyses. By studies at the biochemical level important insights into the destructive modes of action of the phytotoxins have been gained. The strategies for exerting virulence can be manifold: In general, necrotrophic pathogens use toxins to elicit plant cell death and derive nutrition from the dead tissue, whereas biotrophic pathogens rely on living plant tissue. Phytotoxins may interact with a range of cellular targets, alter gene expression or undermine membrane integrity. A number of phytotoxins inhibit the activity of plant enzymes, thereby disrupting the biosynthesis of crucial metabolites. Other fungi interfere with the plants’ physiology by producing plant hormones, such as gibberelin or gibberellic acid (GA, from the rice pathogen Gibberella fujikuroi) [6] or the auxin indole-3-acetic acid (IAA, e.g. from Ustilago maydis, Trichoderma and Moniliophthora perniciosa) [7]. Finally, plant cells may be damaged by the production of reactive oxygen species (ROS) (Figure 1). Some phytotoxins are host-specific on the genus or even on the species level and determine the host range of the fungus by targeting specific enzymes or metabolic pathways [8]. Such host-specific toxins (HSTs) induce pathogenicity only in the host species, where a gene product is the direct or indirect target of the toxin. Nearly all fungi producing HST (i.e. fungi of the genera Alternaria, Cochliobolus, Leptosphaeria, Venturia, Ascochyta and Pyrenophora) belong to the order Pleosporales and appear to have a tendency to lateral gene transfer [1,2]. Finally, it should be noted that already in the primary infection process secondary metabolites can play an important role. Many fungal pathogens penetrate plant leaves from a specialized cell, the appressorium. The appressorium of Magnaporthe oryzae, a rice blast pathogen, is melanized by oxidative polymerization of polyketide precursors and forms an effective barrier to solute movement. Water is entering and an enormous turgor pressure of approximately 80 atmospheres is produced. The resulting force is usually applied to gain entry into plant tissue [9].
Shedding light on plant-damaging photosensitizers Various phytopathogenic fungi produce so-called photosensitizers to generate ROS and thus impair plant cells by induction of apoptosis and damage to membrane lipids. This mechanism has been observed for metabolites sharing a 3,10-dihydroxy-4,9-perylenequinone chromophore, such as cercosporin and elsinochrome (Figure 2). After absorption of light energy, perylenequinones adopt an activated triplet state. The radical can then react with www.sciencedirect.com
Fungal phytotoxins as mediators of virulence Mo¨bius and Hertweck 391
Figure 1
Overview on the cellular targets and the mode of action of several fungal phytotoxins, GDC, glycine decarboxylase; CerS, ceramide synthase, ER, enoyl reductase; HDAC, histone deacetylase complex; NO, nitric oxide; PCD, programmed cell death; PM, plasma membrane.
oxygen to form ROS (O2 , H2O2) and singlet oxygen or induce oxidative lipid decomposition, thus causing major damages to the host cell membrane [10]. Cercosporin is produced by Cercospora species that infect corn, soybean, coffee and other plants. The light-induced damage leads to leakage of nutrients in the intracellular space, thus making them available for fungal hyphae. The fungus protects itself probably via toxin export and quenchers [10]. A polyketide synthase, CTB1, plays a key role in cercosporin biosynthesis in Cercospora nictonianae, as CTB1 mutants cause fewer necrotic lesions on tobacco leaves [11]. Interestingly, expression of the CTB1 gene is highly regulated by light. Disruption of ctb4, a gene coding for a putative membrane transporter, results in decreased cercosporin emission and reduced virulence against tobacco cells [11]. The red-pigmented elsinochromes are produced by Elsinoe fawcettii when exposed to light [12]. Citrus cells, when in contact with the fungus, are rapidly killed whereas www.sciencedirect.com
addition of b-carotene or superoxide dismutase dampens plant cells damage. The polyketide synthase gene Efpks1 essential for elsinochrome production has been identified by gene disruption, and the resulting elsinochrome-negative mutant has a significantly reduced ability to form lesions in lemon leaves [12]. Another light-dependent plant-damaging mechanism has been identified in the context of Ramularia collocygni leaf spot disease on barley. Rubellin D (Figure 2), an anthraquinone derivative, induces peroxidation of alphalinoleic acid in a light dependent manner, predominantly caused by singlet oxygen formation [13]. Furthermore, chlorophyll bleaching was observed in toxin-treated leaves. Interestingly, ferrous ions enhance the rubellin-induced reaction, but repress the action of cercosporin [13].
Protein targeting Epipolythiodioxopiperazines (ETPs) contain a characteristic internal di-sulphide or tri-sulphide bridge (Figure 2). Current Opinion in Plant Biology 2009, 12:390–398
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Figure 2
Structures of apoptosis-inducing phytotoxins victorin and DON, the protein-attacking mycotoxins gliotoxin and sirodesmin (ETP), the actin skeleton targeting cytochalasin B and the photosensitizers cercosporin, elsinochrome A and rubbellin D.
Although the exact mode of action is not yet fully understood, an involvement of this structural feature in protein conjugation [14] or generation of ROS via redox cycling is plausible [14]. Prominent examples of such toxins are sirodesmin PL produced by Leptosphaeria maculans and gliotoxin produced by Trichoderma spp. and Aspergillus fumigatus [15]. A mutant with a disruption of the bimodular NRPS gene (sirP) of L. maculans stalls the production of sirodesmin PL, causes fewer lesions and is half as effective as the wild-type in colonizing stems of Brassica napus (Canola) [15].
Phytotoxins affecting membrane integrity Toxin-mediated inhibition of enzymes involved in lipid biosynthesis is a common strategy applied by phytopathogenic fungi. Important examples are the closely related toxins fumonisin (from Fusarium spp.) and AALtoxin (from Alternaria alternata), which act as sphingosine analogs and inhibit sphinganine–N-acetyltransferase and ceramide synthase (Figure 3) [16,17]. In this way, lipid biosynthesis is hampered, resulting in a perturbed membrane ordering and increased membrane permeability. It Current Opinion in Plant Biology 2009, 12:390–398
should also be noted that toxin-induced changes in ceramide metabolism may have severe consequences for a variety of regulatory processes, as ceramides are involved in intracellular signaling pathways. Gibberella moniliformis, the cause of maize seedling blight, produces fumonisin B1 as one of its virulence factors. Although fumonisin-insensitive maize strains are not resistant to infection, systemic colonization of seedlings is reduced [18]. Cyperin (Figure 3), a diphenyl ether phytotoxin produced by several fungal plant pathogens, interferes with lipid biosynthesis by inhibiting enoyl reductase (ER) [19]. Cyperin is bound to the ER active site by p-p stacking of a phenyl ring and the nicotinamide ring of NAD+. At high concentrations cyperin also blocks protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis [19]. A straightforward way of membrane damage is exerted by the fungus Cercospora beticola using so-called beticolins (Figure 3) or yellow toxins. The polyketides can selfassemble into multimeric structures and form ion www.sciencedirect.com
Fungal phytotoxins as mediators of virulence Mo¨bius and Hertweck 393
Figure 3
Molecules targeting membrane integrity. Sphingosin and phytotoxic analogs AAL-toxin and fumonisin; structure of cyperin, an inhibitor of enoyl reductase and of the pore-forming toxin beticolin 0.
channels in the host membrane (Figure 1). Effects of the pore formation are dramatic loss of solutes, inhibition of ATP-dependent H+-transport and membrane depolarization in various plant species [20].
Taking what’s not given—siderophores in virulence Upon infection – and in particular after penetration – a fungus becomes dependent on the availability of www.sciencedirect.com
nutrients inside of the host. Iron is an essential nutrient and the major redox mediator in cellular processes and thus essential for survival of the pathogen [21]. Iron acquisition is carried out via the secretion and subsequent uptake of low-molecular-weight chelators, so called siderophores, like ferricrocin (A. brassicicola) or triacetylfusarinine C (TAFC, F. gramineum, Figure 4). Mechanisms for iron uptake can be regarded as virulence determinants that lead to iron depletion in the host. Deletion of genes Current Opinion in Plant Biology 2009, 12:390–398
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Figure 4
Structures of molecules causing energy breakdown in plant cells, and the siderophore triacetylfusarinine C.
coding for non-ribosomal peptide synthetases involved in siderophore biosynthesis, for example nps6 (C. heterostrophus) and sid1 (F. gramineum), led to a reduction in virulence to the host plant and to a simultaneous increase in ROS sensitivity in C. heterostrophus. The defects could be compensated by exogenous application of iron [21]. By contrast, the biotroph U. maydis relies on reductive iron uptake rather than on siderophores. Deletion of fer1 and fer2 (encoding a high-affinity iron permease and an iron multicopper oxidase) proved to be much less virulent [22].
calcium has been observed [24]. The toxin is assembled by action of two polyketide synthases (PKSs). One PKS provides the polyketide starter unit for the second PKS, which produces the mature T-toxin molecule. A third biosynthetic gene codes for a decarboxylase (DEC1) [25]. These genes reside in AT-rich DNA that is unique to T-toxin-producing strains (race T). The biosynthetic genes have probably undergone lateral gene transfer, as race T harbors an additional 1.2 Mb of DNA compared with the weakly pathogenic race 0 lacking the Tox1 locus [24].
Energy breakdown
An energy breakdown may also be caused by phytotoxins that affect the integrity of plant plasma membranes (see above), which results in an increased membrane permeability and nutrient leakage, or target H+-ATPase, thus disrupting the electrochemical gradient. It has been shown that fusicoccin, a glycosylated diterpene, augments potassium uptake with concomitant proton extrusion in rice plants, which leads to an increased extracellular acidification [26].
The blockage of ATP-hydrolysis leads to complete energy breakdown in the plant cell. Tentoxin (Figure 4), a host-selective toxin, produced by Alternaria species targets this energy transfer process in the chloroplast. The cyclic tetrapeptide blocks ATP hydrolysis by binding to the surface between the a and b subunits of chloroplast ATPase. The crystal structure of spinach chloroplast F1 with bound tentoxin was solved, and it was shown that a single molecule of the toxin affects ADP release in a non-competitive mechanism [23]. T-toxin, a polyketide synthesized by Cochliobolus heterostrophus, is associated with high virulence on certain genotypes of maize. It selectively targets T cytoplasm mitochondria mediated by the T-urf13 protein, which results in conformational changes and pore formation followed by mitochondrial swelling. Changes in oxidative phosphorylation and respiration as well as leakage of nutrients and Current Opinion in Plant Biology 2009, 12:390–398
Binding of fusicoccin to the plant plasma membrane H+ATPase is mediated by a regulatory protein belonging to the 14-3-3 family. A stable complex with the C-terminus of the H+-ATPase is formed, leading to its permanent activation and irreversible stomata opening. Its mode of action has been elucidated by X-ray crystallographic analysis of the ternary complex of fusicoccin, a plant 14-3-3 protein, and a phosphopeptide derived from the www.sciencedirect.com
Fungal phytotoxins as mediators of virulence Mo¨bius and Hertweck 395
C-terminus of H+-ATPase. Structural analysis and isothermal titration calorimetry indicated that peptide and toxin mutually increase each other’s binding affinity through filling a cavity in the interaction surface [27]. In Arabidopsis thaliana cells, fusicoccin induces an H+ATPase state-independent increase in the extracellular H2O2 level. Measurement on exogenous catalase activity indicated a reduced capability of the cells to degrade H2O2 formed in cell-free media. Apparently an as yet unidentified factor accumulates in the incubation medium of cells treated with fusicoccin, which acts as a noncompetitive catalase inhibitor and is able to reduce the cell’s capacity for H2O2 scavenging [28]. Apart from the mode of action, also the fusicoccin biosynthetic machinery is quite intriguing: PaFS, a multifunctional enzyme of Phomopsis amygdali, plays a key role with prenyltransferase (responsible for condensation of isoprene units) and terpene cyclase (cyclization of C-20 precursor) activity. Both enzymatic functions required for diterpene biosynthesis are normally carried out by two independent enzymes [29].
Strategies to trigger apoptosis The induction of apoptosis, or programmed cell death (PCD) in plants, is a major strategy of phytopathogenic fungi to acquire nutrients from plants. However, the underlying mechanisms can be manifold. Trichothecenes, such as deoxnivalenol (DON), are nonvolatile sesquiterpenoids produced by Fusarium graminearum (Figure 2). DON is a host-specific virulence factor that is frequently found in contaminated cereal crops [30]. In Arabidopsis DON inhibits translation without inducing a plant defense response [30]. The first gene of the trichodiene pathway catalyzes the cyclization of farnesyl pyrophosphate to trichodiene. Disruption of this trichodiene synthase (a terpene cyclasetype enzyme) gene results in a DON-non-producing strain. Conidia of the mutant are still able to infect but disease symptoms are significantly reduced. Mutants deficient in crucial toxin biosynthetic genes exhibited reduced virulence on wheat seedlings but were unaffected with respect to causing disease on barley [31]. These results indicate that these fungal toxins confer host specificity to Fusarium pathogenicity. Notably, wheat infection by the mutants is associated with thickening of the cell wall in the rachis node, a plant induced defense [31]. Self-resistance to DON is conferred by the enzyme trichothecene 3-O-acetyltransferase (Tri101), which transforms DON into 3-acetyldeoxynivalenol (3-ADON). Heterologous expression of Tri101 in rice plants significantly reduced phytotoxic effects [32]. Victorin (Figure 2) is a cyclic pentapeptide produced by the fungus Cochliobolus victoriae that causes Victoria oat blight [33]. The toxin enters mitochondria by a mitowww.sciencedirect.com
chondrial permeability transition (MPT) and binds to Pprotein of the mitochondrial matrix, a subunit of the glycine decarboxylase complex (GDC, part the photorespiratory cycle). After incubation with victorin a rapid response is observed, in particular DNA laddering, lipid oxidation and cleavage of RUBISCO, followed by the inhibition of photorespiration [34]. Recently, victorininsensitive mutants were isolated, with resistance conferred to specifically by a mutation in liv1 (locus of insensitivity to victorin1). The gene product is thioredoxin h5 (ATTRX5), a member of a large family of disulphide oxidoreductases [34]. Sensitivity to the toxin in Arabidopsis thaliana relies on the gene lov1 that codes for a coiledcoil–nucleotide binding site leucine-rich repeat protein. Ironically, lov1 is a member of the NBS–LRR resistance gene family [35]. Finally, PCD can also be induced by the above-mentioned fusiccocin. An apoptotic-like form of PCD that involves typical apoptotic characteristics like chromatin condensation, Cytochrome c release and DNA laddering and is inhibited by cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria [36].
Breaking the cell’s bones—destroying the actin skeleton Disaggregation of the cytoskeleton can ultimately also lead to PCD and is another plant-damaging effect caused by fusiccocin (see above). Actin depolymerization seems to be mediated by induction of NO production and can be decreased by the addition of NO scavengers or actin stabilizing drugs [36]. By contrast, toxins belonging to the family of cytochalasans (gr.: kytos – cell, and chalasis – relaxation) specifically bind to actin filaments, thus blocking cytokinesis, while mitosis remains unaffected [37]. This structurally diverse group of polyketide–amino acid hybrids includes, among others, cytochalasin A, cytochalasin B (phomin), and chaetoglobosins, which are particularly active representatives (Figure 2) [37]. The molecular basis for chaetoglobosin A and C biosynthesis was elucidated in Penicillium expansum. A PKS–NRPS hybrid synthetase (CheA) assembles the linear polyketide–amino acid backbone that finally undergoes a Diels–Alder reaction to yield the tricyclic framework [38]. Interestingly, related genes coding for PKS-NRPS hybrids are present in the genome [3] of Magnaporthe oryzae. The M. oryzae ACE1 avirulence gene encodes a putative hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), with the structure of the biosynthetic product not yet elucidated. Thus, it is not clear whether the product of this gene will affect the actin cytoskeleton. Expression of ACE1 occurs early during infection and coincides with penetration of the cuticle [39]. Current Opinion in Plant Biology 2009, 12:390–398
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To sight the core-transcription factors, epigenetic modifiers and antimitotic agents Manipulation of the replication machinery and altering gene expression profiles are other important effects mediated by secreted fungal compounds. An important example is Ustilago maydis, a ubiquitous biotrophic fungus and maize pathogen. While its complete genome sequence has revealed only few secondary biosynthetic genes [4], it was found that auxin (indole-3-acetic acid, IAA) is produced. Since the concentration of auxin in U. maydis induced tumors is significantly higher (up to 20fold) in tumor tissue than in the surrounding plant tissues, fungal production of this plant growth hormone was implicated in tumor induction [40]. According to the current model IAA is involved in targeted protein degradation of the Aux/IAA transcriptional repressors via the auxin receptor TIR1 and thereby regulates transcription [41]. However, a quadruple mutant that is unable to produce IAA in culture was unaffected in tumor induction, suggesting the elevated IAA levels in tumor tissue are likely to result from fungus-induced changes in plant hormone levels [40]. As the biotrophic lifestyle requires efficient protection against plant defense reactions. Notably, in the genome of U. maydis a gene coding for a Yap1-related protein has been identified that serves as the central regulator providing S. cerevisiae. In U. maydis this transcription factor is involved in detoxification of plant produced ROS and fundamental for full virulence [42]. Another growth-regulating phytohormone is the already mentioned gibberellin (GA) that belongs to a group of diterpenoid acids responsible for stem elongation and seed germination [43]. It was first isolated from the pyhtopathogenic fungus Gibberella fujikuroi, which causes the rice plant disease Bakanae whose most prominent characteristics are strongly elongated seedlings [6]. The plant and fungal biosynthetic pathways differ only in the
last steps [6]. Recently the GA receptor GID1 has been identified in Arabidopsis. GID1 is located in the nucleus and enables degradation of a repressor bound to GAdependent transcription factors through ubiquitin ligation and leads to gene transcription [43]. Another mechanism to alter gene expression involves epigenetic modification. This strategy is employed by Cochliobolus carbonum, a fungus that is highly virulent on certain maize genotypes. C. carbonum produces HC-toxin, a cyclic tetrapeptide containing D-amino acids (Figure 5), and is highly virulent on certain genotypes of maize. Production of the host-selective compound is under the control of a complex locus (tox2) coding for HTS1 (HC-toxin synthetase), an NRPS [44]. The putative HCtoxin efflux carrier encoded by toxA is probably involved in a self-protection mechanism. The toxin is an inhibitor of histone deacetylases (HDACs) leading to hyperacetylation and thereby to changes in gene expression in the plant. In vitro kinetic studies revealed that the inhibition is uncompetitive and reversible [45]. HC-toxin stimulates the uptake of organic and inorganic molecules such as nitrate into the maize roots. Resistant maize plants carry the Hm1 resistance gene that encodes an HC-toxin reductase (HCTR) [45]. Chromosomal segregation is of vital importance for cell division and growth, and its inhibition is an effective strategy of a pathogenic organism. The antimitotic agent rhizoxin (Figure 5), a macrocyclic polyketide, is known as the virulence factor of the rice seedling blight fungus Rhizopus microsporus. Rhizoxin binds to b-tubulin, thus preventing heterodimerization with a-tubulin and consequently the formation of microtubules [46]. Only recently, it was found that the toxin is not produced by the fungus, but by bacterial endosymbionts that reside within the fungal cytosol [46]. Isolation and cultivation of the symbionts clearly demonstrated that the toxin complex is produced by the endofungal bacterium
Figure 5
Molecules targeting processes in the nucleus; phytotoxins acting as epigenetic modifier (HC-toxin) or blocking mitosis (rhizoxin). Current Opinion in Plant Biology 2009, 12:390–398
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Fungal phytotoxins as mediators of virulence Mo¨bius and Hertweck 397
Burkholderia rhizoxinica [47]. Furthermore, the molecular basis for rhizoxin production was elucidated by sequencing a gene cluster coding for a modular PKS–NRPS assembly line in the bacterial genome [48]. A specific mutation of b-tubulin confers resistance to the fungal host and is probably a precondition for the establishment of the symbiosis [49]. This is the first described case where a phytotoxin employed by a fungus is actually produced by endosymbionts. Here, the pathogenic relationship between the plant and the infecting fungus is extended to a tripartite system including a symbiotic alliance of fungus and bacterium. The fungus benefits from plant nutrients but is itself dependent on bacteria for toxin production and even for reproduction as spore formation can only occur in the presence of the bacterial symbionts [50].
Conclusion In conclusion, phytopathogenic fungi employ an array of strategies to distress, weaken or kill the host plant in order to gain access to nutrients. The captivating structural and mechanistic diversity of the toxins teaches us a lesson on the complexity of pathogenic relationships—up to the point where a metabolically lean fungus hosts a bacterium for toxin production. Understanding toxin biosynthesis pathways and their regulation, the modes of action and how this relates to fungal virulence will not only help to gain new insights into cellular processes in general but is also a stepping stone to develop ways to protect plants from fungal infections. With the help of advanced genomics, proteomics and analytical skills we will soon understand more about phytotoxins as mediators of plant virulence.
Acknowledgment The authors are grateful for financial support of original research in this area by the Jena School for Microbial Communication (JSMC).
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enzymatic functions that are involved in the production of secondary metabolites will greatly profit from the genome sequence of U. maydis. Some of the predicted genes are suggested to be subject to high genetic and genomic variation. 5.
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