a b s t r a c tBotrydial (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) 2 Cys 6 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.

No abstract

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Atf1-homologous basic region leucine zipper (bZIP) transcription factors are known to act downstream of the stress-activated mitogen-activated protein kinase (SAPK) cascade in mammals, as well as in several fungi; they regulate the transcription of genes involved in the general stress response. Functional analyses of BcAtf1 in Botrytis cinerea show that it is also connected to the SAPK BcSak1, as it shares several stress response target genes. However, Δbcatf1 mutants are not hypersensitive to osmotic or oxidative stress, as are Δbcsak1 mutants. Both BcSak1 and BcAtf1 are regulators of differentiation, but their roles in these processes are almost inverse as, in contrast with Δbcsak1, Δbcatf1 mutants are significantly impaired in conidia production and do not differentiate any sclerotia. They show extremely vigorous growth in axenic culture, with a thick layer of aerial hyphae and a marked increase in colonization efficiency on different host plants and tissues. In addition, the sensitivity to cell wall-interfering agents is increased strongly. Microarray analyses demonstrate that the loss of BcAtf1 leads to extensive transcriptional changes: apart from stress response genes, the expression of a broad set of genes, probably involved in primary metabolism, cell wall synthesis and development, is affected by BcAtf1. Unexpectedly, BcAtf1 also controls secondary metabolism: the mutant contains significantly elevated levels of phytotoxins. These data indicate that BcAtf1 controls a diversity of cellular processes and has broad regulatory functions.

Trichothecenes are sesquiterpenoid mycotoxins produced mainly by Fusarium species. Harzianum A (HA), a non-phytotoxic trichothecene produced by Trichoderma arundinaceum, has recently been found to have antagonistic activity against fungal plant pathogens and to induce plant genes involved in defense responses. In the present work, we have shown that disruption of the T. arundinaceum tri5 gene, which encodes a terpene synthase, stops the production of HA, alters the expression of other tri genes involved in HA biosynthesis, and alters the expression of hmgR, dpp1, erg9, erg1, and erg7, all genes involved in terpene biosynthetic pathways. An increase in the level of ergosterol biosynthesis was also observed in the tri5 disrupted transformant in comparison with the wild type strain. The loss of HA also resulted in a drastic reduction of the biocontrol activity of the transformants against the phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani. Finally, the effect of tri5 gene disruption on the regulation and balance of intermediates in terpene biosynthetic pathways, as well as the hypothetical physiological role of trichothecenes, both inter- and intracellularly, on regulation and biocontrol, are discussed.