Paul Tudzynski

Botrytis cinerea: the cause of grey mould disease.

INTRODUCTION Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle attacking over 200 crop hosts worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi. TAXONOMY Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botryotinia. HOST RANGE AND SYMPTOMS Over 200 mainly dicotyledonous plant species, including important protein, oil, fibre and horticultural crops, are affected in temperate and subtropical regions. It can cause soft rotting of all aerial plant parts, and rotting of vegetables, fruits and flowers post-harvest to produce prolific grey conidiophores and (macro)conidia typical of the disease. PATHOGENICITY B. cinerea produces a range of cell-wall-degrading enzymes, toxins and other low-molecular-weight compounds such as oxalic acid. New evidence suggests that the pathogen triggers the host to induce programmed cell death as an attack strategy. Resistance: There are few examples of robust genetic host resistance, but recent work has identified quantitative trait loci in tomato that offer new approaches for stable polygenic resistance in future. USEFUL WEBSITES http://www.phi-base.org/query.php, http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html, http://urgi.versailles.inra.fr/projects/Botrytis/, http://cogeme.ex.ac.uk.

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Botrytis : biology, pathology and control

Preface.- Acknowledgements.- Contributors.- 1: Botrytis spp. and diseases they cause in agricultural systems - an introduction Yigal Elad, Brian Williamson, Paul Tudzynski and Nafiz Delen.- 1 Introduction. 2. Geographical and ecological occurrence. 3. Variability and adaptability. 4. Quiescent, restricted and aggressive infection. 5. Molecular basis of host-parasite interactions. 6. References. 2: The Ecology of Botrytis on Plant Surfaces Gustav Holz, Sonja Coertze and Brian Williamson.- 1. Introduction. 2. Survival. 3. Inoculum production and dispersal. 4. Growth on plant surfaces. 5. Infection pathways on diverse plant organs. 6. Conclusion. 7. References. 3: Taxonomy and Genetic Variation of Botrytis and Botryotinia Ross E. Beever and Pauline L. Weeds.- 1. Introduction. 2. Taxonomy. 3. Botrytis cinerea. 4. Genetics of other species of Botrytis. 5. The future. 6. Acknowledgements. 7. References. 4: Approaches to Molecular Genetics and Genomics of Botrytis Paul Tudzynski and Verena Siewers.- 1. Introduction. 2. Generation of transgenic Botrytis strains. 3. Unbiased gene cloning systems. 4. Perspectives. 5. Acknowledgements. 6. References. 5: Morphology and Cellular Organisation in Botrytis Interactions with Plants Klaus B. Tenberge.- 1. Introduction. 2. Cytology and ultrastructure of Botrytis. 3. Imaging of infection. 4. Host response. 5. Conclusions. 6. Acknowledgements. 7. References. 6: Signalling in Botrytis cinerea Bettina Tudzynski and Christian Schulze Gronover.- 1. Introduction. 2. Ga subunits of hetrotrimeric G proteins. 3. cAMP signalling pathway. 4. MAP kinase pathways. 5. Genes of the Ras superfamily. 6. Calcineurin/cyclophilin A signalling. 7. Putative transmembrane receptor proteins. 8. Two-component signal transduction genes in Botrytis cinerea. 9. Further protein kinase encoding geneswith unknown function. 10. Conclusion. 11. References. 7: Extracellular Enzymes and Metabolites Involved in Pathogenesis of Botrytis Ilona Kars and Jan A.L. van Kan.- 1. Introduction. 2. Penetration of the host surface. 3. Killing of host cells. 4. Conversion of host tissue into fungal biomass. 5. Other enzymes potentially involved in pathogenesis. 6. Concluding remarks. 7. Acknowledgements. 8. References. 8: Botrytis cinerea Perturbs Redox Processes as an Attack Strategy in Plants Gary D. Lyon, Bernard A. Goodman and Brian Williamson.- 1. Introduction. 2. Hydrogen peroxide and other AOS. 3. Low molecular mass antioxidant molecules. 4. Perturbation of free radical chemistry as a result of Botrytis infection. 5. Production of oxalic acid. 6. Dynamics of iron redox chemistry. 7. Regulation of plant enzymes. 8. Botrytis-derived enzymes. 9. Generation of lipid peroxidation products. 10. Host signalling and programmed cell death. 11. Fungus-derived metabolites. 12. Conclusion. 13. Acknowledgements. 14. References. 9: Plant Defence Compounds against Botrytis Infection Peter van Baarlen, Laurent Legendre and Jan A.L. van Kan.- 1. Introduction. 2. Antimicrobial secondary metabolites. 3. Tolerance of Botrytis to antifungal metabolites. 4. Structural barriers and cell wall modifications. 5. Pathogenesis-related proteins. 6. Concluding remarks. 7. Acknowledgements. 8. References. 10: Phytohormones In Botrytis-Plant Interactions Amir Sharon, Yigal Elad, Radwan Barakat and Paul Tudzynski.- 1. Introduction. 2. Biosynthesis of plant hormones by B. cinerea. 3. Effect of plant hormones on B. cinerea and on disease development. 4. Conclusions. 5. Acknowledgement. 6. References. 11: Detection, Quantification and Immunolocalisation of Botrytis species Frances M. Dewey (Molly) and David Yohalem.- 1. Introduction. 2. Classical plating out method. 3. Immu

Reactive Oxygen Species in Phytopathogenic Fungi: Signaling, Development, and Disease

Reactive oxygen species (ROS) play a major role in pathogen-plant interactions: recognition of a pathogen by the plant rapidly triggers the oxidative burst, which is necessary for further defense reactions. The specific role of ROS in pathogen defense is still unclear. Studies on the pathogen so far have focused on the importance of the oxidative stress response (OSR) systems to overcome the oxidative burst or of its avoidance by effectors. This review focuses on the role of ROS for fungal virulence and development. In the recent years, it has become obvious that (a) fungal OSR systems might not have the predicted crucial role in pathogenicity, (b) fungal pathogens, especially necrotrophs, can actively contribute to the ROS level in planta and even take advantage of the hosts response, (c) fungi possess superoxide-generating NADPH oxidases similar to mammalian Nox complexes that are important for pathogenicity; however, recent data indicate that they are not directly involved in pathogen-host communication but in fungal differentiation processes that are necessary for virulence.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Functional analysis of H2O2-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable

SUMMARY The oxidative burst, a transient and rapid accumulation of reactive oxygen species (ROS), is a widespread defence mechanism of higher plants against pathogen attack. There is increasing evidence that the necrotrophic fungal pathogen Botrytis cinerea itself generates ROS, and that this capability could contribute to the virulence of the fungus. Two potential H(2)O(2)-generating systems were studied with respect to their impact on the interaction of B. cinerea and its host plant Phaseolus vulgaris. A Cu-Zn-superoxide dismutase gene (bcsod1) and a putative glucose oxidase gene (bcgod1) were cloned and characterized, and deletion mutants were created using a gene-replacement methodology. Whereas the Deltabcgod1-mutants displayed normal virulence on bean leaves, the Deltabcsod1 mutants showed a significantly retarded development of lesions, indicating that the Cu-Zn SOD-activity is an important single virulence factor in this interaction system. Whether dismutation of (fungal or host) superoxide, or generation of H(2)O(2) (or both), are important for pathogenesis in this system remains to be elucidated.

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

Abstract A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Chapter 2 Ergot Alkaloids – Biology and Molecular Biology☆

Publisher Summary This chapter discusses the biology and molecular biology of ergot alkaloids (EA). The EA are among the most important natural pharmaceuticals and toxins in human history. The EA are characterized by the tetracyclic ergoline ring system or by related tricyclic alkaloids open between N(6) and C(7) (ergoline numbering). They are categorized as clavines, lysergic acid (1) and its simple amides, and ergopeptines. The distribution of organisms possessing EA appears disjointed, including two orders of fungi and three plant families. The EA-producing fungi are in the Eurotiales and Hypocreales, two distantly related orders within the phylum Ascomycota. The main ecological roles of EA in nature are probably to protect the fungi from consumption by vertebrate and invertebrate animals. The EA produced by plant-symbiotic fungi (such as epichloe¨ endophytes) may protect the fungus by protecting the health and productivity of the host, which may otherwise suffer excessive grazing by animals. The EA, at levels typical of plants bearing these symbionts, can negatively affect the health of large mammals as well herbivorous insects. Some clavines have substantial anti-bacterial properties, which might protect the fungus and, in some cases, their host plants from infection.

The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea.

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding alpha subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the G alpha(i) class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.

NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea.

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67(phox), a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of DeltabcnoxR mutants is identical to that of DeltabcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.