Akihiko Ichiishi

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT 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.

Putative Homologs of SSK22 MAPKK Kinase and PBS2 MAPK Kinase of Saccharomyces cerevisiae Encoded by os-4 and os-5 Genes for Osmotic Sensitivity and Fungicide…

We cloned and characterized Neurospora NcSSK22 and NcPBS2 genes, similar to yeast SSK22 mitogen-activated protein (MAP) kinase kinase kinase and the PBS2 MAP kinase kinase genes, respectively. Disruptants of the NcSSK22 gene were sensitive to osmotic stress and resistant to iprodione and fludioxonil. Their phenotypes were similar to those of osmotic-sensitive (os) mutants os-1, os-2, os-4, and os-5. The os-4 mutant strain transformed with the wild-type NcSSK22 gene grew on a medium containing 4% NaCl and was sensitive to iprodione and fludioxonil. In contrast, the NcPBS2 gene complemented the osmotic sensitivity and fungicide resistance of the os-5 mutant strain. We sequenced the NcPBS2 gene of the os-5 mutant strain (NM216o) and found five nucleotides deleted within the kinase domain. This result suggests that the gene products of os-4 and os-5 are components of the MAP kinase cascade, which is probably regulated upstream by two-component histidine kinase encoded by the os-1/nik1 gene.

An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast

Three groups of fungicides (phenylpyrroles, dicarboximides, aromatic hydrocarbons) are effective against filamentous fungi. The target of these fungicides is the osmotic stress signal transduction pathway, which is dependent on the Os-1 family of two-component histidine kinases. These fungicides usually have no fungicidal effect on the yeast Saccharomyces cerevisiae. In this report, we found that expression of Hik1, an Os-1 orthologue from rice blast fungus, can confer fungicide-sensitivity to yeast. This requires both the histidine kinase and the response regulator domains of Hik1. Analysis of yeast mutants indicated that this sensitivity is Hog1- and Ssk1-dependent. In addition, our studies revealed an interaction between Hik1 and Ypd1. These observations suggest that Hik1 is a direct target of the fungicides or is a mediator of fungicide action and that the fungicidal effect is transmitted to the Hog1 pathway via Ypd1.

Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

Effects of Iprodione and Fludioxonil on Glycerol Synthesis and Hyphal Development in Candida albicans

We investigated the effects of iprodione and fludioxonil on the pathogenic yeast Candida albicans. Growth of the wild-type IFO1385 strain of C. albicans was inhibited by both fungicides, while Saccharomyces cerevisiae was basically unaffected by them even at a concentration of 25 μg/ml. Both fungicides stimulated glycerol synthesis in C. albicans but not in S. cerevisiae. The antioxidant α-tocopherol acetate and the cytochrome P-450 inhibitor piperonyl butoxide antagonized the fungitoxicity of iprodione and fludioxonil in C. albicans. It is known that mutations within the histidine kinase NIK1/OS-1 gene confer resistance to iprodione and fludioxonil in Neurospora crassa, while the fungicide-insensitive S. cerevisiae has only one histidine kinase SLN1 gene in its genome. In contrast, C. albicans has three histidine kinase genes, namely CaSLN1, CaNIK1/COS1, and CaHK1, the null mutants of which were found to impair the hyphal formation. Iprodione and fludioxonil were found to suppress filamentation when the IFO1385 strain was incubated on a solid medium containing fetal bovine serum. These observations suggest that iprodione and fludioxonil interfere with the CaNIK1/COS1 signal transduction pathway, resulting in glycerol synthesis stimulation and the inhibition of hyphal formation.

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (368Val to Phe, 369Gln to His, and 447Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (369Gln to Pro and 373Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.

An AP-1-Like Transcription Factor, NAP-1, Regulates Expression of the Glutathione S-Transferase and NADH:Flavin Oxidoreductase Genes in Neurospora crassa

AP-1-like transcription factors play crucial roles in oxidative stress responses in yeast and filamentous fungi. The deletion of an AP-1-like transcription factor gene, nap-1, in Neurospora crassa slightly increased its sensitivity to oxidative stressors, including menadione. Microarray and quantitative real-time reverse transcriptase-PCR analyses were employed to identify menadione-inducible genes (migs) and the roles of NAP-1 in their regulation. N. crassa migs include three putative glutathione S-transferase genes and two NADH:flavin oxidoreductase genes, orthologs of OYE2 and OYE3, both of which play roles in menadione tolerance in Saccharomyces cerevisiae. Menadione induced nuclear localization of NAP-1, and oxidative upregulation of many of migs were NAP-1 dependent. Genes for a thioredoxin, a glutathione reductase, and a glutathione peroxidase were slightly upregulated by the chemical only in the wild-type strain, suggesting that NAP-1 is involved in their oxidative induction and probably dose not contribute to high-level constitutive expressions of such genes.

OS-2 Mitogen Activated Protein Kinase Regulates the Clock-Controlled Gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.

Deletion and expression analysis of beta-(1,3)-glucanosyltransferase genes in Neurospora crassa.

GPI(glycosylphosphatidylinositol)-anchored beta-(1,3)-glucanosyltransferases play an active role in cell wall biosynthesis in fungi. Neurospora crassa has 5 putative beta-(1,3)-glucanosyltransferase genes, namely, gel-1, gel-2, gel-3, gel-4, and gel-5, in its genome. Among them, the gel-3 gene is constitutively expressed at the highest level in growing hyphae, whereas gel-1 is expressed at the lowest level. The gel-3 deletion mutant displayed slow growth, while other gel gene disruptants exhibited normal growth. Although no gel gene disruption affected pH sensitivity and fertility, all Δgel mutants were resistant to cell wall degradation enzymes. Micafungin, a beta-(1,3)-glucan synthase inhibitor, induced gel-4 expression in the wild-type and 2 MAP kinase mutants mak-1 and mak-2 strains. In contrast, fludioxonil, an activator of OS-2 MAP kinase, strongly induced the gel-1 gene in the wild-type strain. Its induction was nearly abolished in the os-2 and in the atf-1/asl-1 mutant. These suggested that GEL-3 is a major factor in mycelial growth, while GEL-1 and GEL-4 may play important roles in cell wall remodeling in response to stress conditions or cell wall damage, respectively.

Histidine Kinase Inhibitors

Dicarboximides and phenylpyrroles have been mainly used to control diseases caused by fungal strains that belong to the genera Botrytis, Sclerotinia, Monilinia, and Alternaria. Both types of fungicides overactivate Hog-like mitogen-activated protein kinases in the osmotic signal transduction pathway and result in cell death. Cross-resistance among dicarboximides, phenylpyrroles, and aromatic hydrocarbons has been observed in most laboratory Botrytis cinerea-resistant mutants, which are generally hyperosmotic sensitive. However, such resistant strains have rarely been isolated from the fields. All dicarboximide-resistant field isolates contained point mutations in a putative osmosensor histidine kinase BcOS1/Daf1, did not show cross-resistance to phenylpyrroles, and were insensitive to osmotic stress. In contrast, Alternaria field-resistant strains carried various mutations, including null mutations, in their osmosensor histidine kinase genes. The introduction of several new fungicides against B. cinerea, such as anilinopyrimidine fungicides, fenhexamid, QoIs, and succinate dehydrogenase inhibitors, reduced the use of dicarboximides, thereby reducing the populations of dicarboximide-resistant strains. However, several types of multidrug resistance strains, in which efflux pumps are activated, have emerged. Gain-of-function mutations of the transcription factor Mrr1, which leads to an overexpression of the ATP-binding cassette transporter AtrB, confers reduced sensitivities to some fungicides, including fludioxonil and cyprodinil. In addition, strains that overexpress the major facilitator superfamily transporter mfsM2 by promoter rearrangements lead to reduced sensitivities to iprodione, fenhexamid, and cyprodinil. Therefore, in addition to target modifications of BcOS1, multidrug resistance caused by the overexpression of drug transporters is another resistance mechanism in B. cinerea against dicarboximides and phenylpyrroles.