Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.

No abstract

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme.

Terpendole E is the first natural product inhibitor of kinesin Eg5. Because terpendole E production is unstable, we isolated and analyzed the terpendole E biosynthetic gene cluster, which consists of seven genes encoding three P450 monooxygenases (TerP, TerQ, and TerK), an FAD-dependent monooxygenase (TerM), a terpene cyclase (TerB), and two prenyltransferases (TerC and TerF). Gene knockout and feeding experiments revealed that terpendole E is a key intermediate in terpendole biosynthesis and is produced by the action of the key enzyme TerQ from paspaline, a common biosynthetic intermediate of indole-diterpenes. TerP converts terpendole E to a downstream intermediate specific to terpendole biosynthesis and converts paspaline to shunt metabolites. We successfully overproduced terpendole E by disrupting the terP gene. We propose that terpendole E is a key biosynthetic intermediate of terpendoles and related indole-diterpenes.

Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.