Genetic and molecular basis of botrydial biosynthesis: connecting cytochrome P450-encoding genes to biosynthetic intermediates

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G + C/A + T-equilibrated regions that contain the Bcbot genes alternate with A + T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the DBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle

Genetic Evidence for a Link Between Glycolysis and DNA Replication

BACKGROUND: A challenging goal in biology is to understand how the principal cellular functions are integrated so that cells achieve viability and optimal fitness in a wide range of nutritional conditions. METHODOLOGY/PRINCIPAL FINDINGS: We report here a tight link between glycolysis and DNA synthesis. The link, discovered during an analysis of suppressors of thermosensitive replication mutants in bacterium Bacillus subtilis, is very strong as some metabolic alterations fully restore viability to replication mutants in which a lethal arrest of DNA synthesis otherwise occurs at a high, restrictive, temperature. Full restoration of viability by such alterations was limited to cells with mutations in three elongation factors (the lagging strand DnaE polymerase, the primase and the helicase) out of a large set of thermosensitive mutants affected in most of the replication proteins. Restoration of viability resulted, at least in part, from maintenance of replication protein activity at high temperature. Physiological studies suggested that this restoration depended on the activity of the three-carbon part of the glycolysis/gluconeogenesis pathway and occurred in both glycolytic and gluconeogenic regimens. Restoration took place abruptly over a narrow range of expression of genes in the three-carbon part of glycolysis. However, the absolute value of this range varied greatly with the allele in question. Finally, restoration of cell viability did not appear to be the result of a decrease in growth rate or an induction of major stress responses. CONCLUSIONS/SIGNIFICANCE: Our findings provide the first evidence for a genetic system that connects DNA chain elongation to glycolysis. Its role may be to modulate some aspect of DNA synthesis in response to the energy provided by the environment and the underlying mechanism is discussed. It is proposed that related systems are ubiquitous

Régulation spécifique des clusters botrydial et acide botcinique chez Botrytis cinerea

Botrytis cinerea est l’agent pathogène responsable de la pourriture grise qui touche plus de 200 espèces végétales. Parmi les facteurs de virulence identifiés chez ce champignon ascomycète figurent deux toxines (et leurs dérivés respectifs) ayant un rôle redondant dans la nécrotrophie. Il s’agit du sesquiterpène botrydial (BOT) et du polycétide acide botcinique (BOA). Les gènes responsables de leur synthèse sont agencés en clusters au sein du génome comme dans la plupart des cas pour les gènes du métabolisme secondaire chez les champignons. Des études précédentes ont permis de mettre en évidence la régulation de ces clusters par des voies de transductions conservées de type MAP kinases ainsi que par des facteurs de transcription globaux comme le facteur de transcription calcineurine-dépendant BcCrz1. De plus, un fort lien entre l’expression de ces gènes du métabolisme secondaire et le développement en réponse à la lumière a été démontré chez l’agent de la pourriture grise, notamment par la caractérisation des membres du complexe Velvet BcVel1 et BcLae1. Des données de régulation globale sont donc disponibles pour les gènes Bcbot et Bcboa mais leurs régulateurs directs n’ont pas été caractérisés. D’après de nombreux exemples de la littérature, lorsqu’un gène codant un facteur de transcription se trouve au sein d’un cluster du métabolisme secondaire, la protéine produite régule spécifiquement les gènes du cluster. Parmi les gènes Bcboa, Bcboa13 est prédit pour coder un facteur de transcription de type Zn(II)2Cys6. D’autre part, une nouvelle version du génome de B. cinerea a permis de compléter la liste des gènes putatifs du cluster BOT et de mettre en évidence à proximité des gènes Bcbot1 à 5 un gène (Bcbot6) prédit pour coder également un FT de type Zn(II)2Cys6. Dans le cadre de notre étude, la caractérisation fonctionnelle de BcBot6 et BcBoa13 est réalisée. Notre hypothèse est que ces facteurs de transcription sont des régulateurs spécifiques de leur cluster. Les données acquises jusqu’ici via la création de mutants et des analyses d’expression par RT-qPCR confirment cette hypothèse. Afin d’étudier la capacité de BcBot6 et BcBoa13 d’intervenir dans des interactions directes avec les promoteurs Bcbot et Bcboa, une approche par simple hybride est actuellement réalisée. Finalement, la banque de facteurs de transcription dont nous disposons sera criblée pour identifier des régulateurs de Bcbot6 et Bcboa13 afin de faire le lien avec les cascades d’activation déjà̀ connues

The VELVET Complex in the Gray Mold Fungus Botrytis cinerea: Impact of BcLAE1 on Differentiation, Secondary Metabolism, and Virulence

Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light- dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia for- mation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulenc

BOA13, un facteur de transcription qui régule la synthèse d’acide botcinique et d’autres fonctions impliquées dans la nécrotrophie

Botrytis cinerea est le champignon nécrotrophe responsable de la pourriture grise qui affecte plus de 200 espèces végétales. Cet agent phytopathogène possède un fort potentiel de synthèse de métabolites secondaires (MSs). Le rôle de plusieurs de ces métabolites, dont le polycétide acide botcinique, a été mis en évidence dans la mort des cellules de l’hôte et dans la colonisation de ses tissus. Les mécanismes moléculaires qui régulent l’expression des gènes codant ces MSs restent cependant jusqu’ici méconnus et leur étude présente donc un réel intérêt. Les gènes responsables de la synthèse de l’acide botcinique sont organisés en cluster (BOA) et l’un d’entre eux code un facteur de transcription (BOA13). L’analyse transcriptomique globale du mutant Δboa13 a permis de mettre en exergue le rôle régulateur attendu du facteur de transcription (FT) sur les gènes du cluster BOA mais aussi de façon inattendue sur des gènes impliqués dans d’autres aspects de la nécrotrophie (production d’enzymes de dégradation des parois végétales, protéases, transporteurs membranaires et réponse au stress oxydatif). Ce résultat est original puisqu’il révèle une régulation par BOA13 de gènes qui ne sont pas impliqués dans le métabolisme secondaire. Les travaux en cours visent (i) à identifier les motifs de fixation de BOA13 et (ii) à déterminer si ce FT interagit directement avec les promoteurs des gènes candidats. Pour cela, ils consistent dans un premier temps à réaliser une analyse en microscopie à fluorescence des promoteurs entiers et tronqués fusionnés à la GFP au sein de transformants de B. cinerea. Le niveau d’intensité du signal de fluorescence permettra de mettre en évidence l’implication des régions d’intérêt dans la fixation de FTs. Ensuite, l’outil simple-hybride développé au sein du laboratoire sera utilisé pour mettre en évidence l’interaction physique possible entre BOA13 et les motifs validés lors de l’étape de microscopie. L’ensemble des FTs de la banque simple-hybride sera aussi criblé afin de vérifier si certains d’entre eux interagissent avec les motifs étudiés. L’ensemble de ces résultats nous permettra d’approfondir les connaissances relatives à la régulation de la synthèse de toxines chez le champignon nécrotrophe modèle B. cinerea

Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaEBs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaEBs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex. We characterize their activities and analyse the functional significance of their interactions using primase, helicase and primer extension assays, and a ‘stripped down’ reconstituted coupled assay to investigate the coordinated displacement of the parental duplex DNA at a replication fork, synthesis of RNA primers along the lagging strand and hand-off to DnaEBs. The DnaG– DnaEBs hand-off takes place after de novo polymerization of only two ribonucleotides by DnaG, and does not require other replication proteins. Furthermore, the fidelity of DnaEBs is improved by DnaC and DnaG, likely via allosteric effects induced by direct protein–protein interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaEBs. We conclude that de novo RNA primer synthesis by DnaG and initial primer extension by DnaEBs are carried out by a lagging strand–specific subcomplex comprising DnaG, DnaEBs and DnaC, which stimulates chromosomal replication with enhanced fidelity

Analysis of the molecular dialogue between gray mold (Botrytis cinerea) and grapevine (Vitis vinifera) reveals a clear shift in defense mechanisms during berry ripening

International audienceMature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus development. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes up-regulated during infection of MB are enriched in functional categories related to necrotrophy such as degradation of plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation and detoxification. Quantitative-PCR on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but stops at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathways switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin and cell-wall strengthening. On the contrary, in infected MB the jasmonate-dependent pathway was activated which did not stop the fungal necrotrophic process

The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster. Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation. Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8 -ol