Mitsuya Tsuda

Cloning and characterization of the histidine kinase gene Dic1 from Cochliobolus heterostrophus that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.

Molecular phylogeny ofNothofagus (Nothofagaceae) based on theatpB-rbcL intergenic spacer of the chloroplast DNA

The genusNothofagus is distributed in the Southern Hemisphere from South America to Oceania, and its distribution has been assumed to be formed by continental drift by means of Gondwana break-up during the Mesozoic era. The phylogeny of the genus was elucidated by the sequences ofatpB-rbcL intergenic spacer of cpDNA for the better understanding of its evolution and biogeography. The phylogeny ofNothofagus corresponded completely to the pollen morphology which recognizes four pollen types in extant species, and agrees well with the taxonomic system of Hill and Read (1991) although there, the subgenusNothofagus showed in unresolved polytomy. The topology of the phylogenetic tree reveals that subgenusLophozonia was derived first, and thenFuscospora, Nothofagus andBrassospora. Species from South America and New Zealand were assigned to each cluster according to their pollen morphology. Therefore, diversification ofNothofagus should have already proceeded at the subgenus level before the completion of Gondwana break-up Tropical species distributed in New Guinea and New Caledonia whose evolutionary history has been controversial were revealed to be a derived group. All five New Caledonian species formed a monophyletic group with very few sequence divergences in the intergenic spacer of cpDNA, thus showing rapid adaptive radiation in the island. Evolutionary trends of several morphological traits ofNothofagus are discussed. The evolution of valve number of cupules, number of nuts per cupule, and habit of leaf-fall (evergreen or deciduous) which are diversified in the genus, were revealed as having occurred several times as the result of convergence.

Changes in the Content and Biosynthesis of Phytoalexins in Banana Fruit

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-13C] and [2-13C]cinnamic acids, and [2-13C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4′-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.

RFLP analysis for species separation in the genera Bipolaris and Curvularia

Restriction fragment length polymorphism (RFLP) of the total DNA ofBipolaris andCurvularia species was analysed using arbitrarily chosen genomic clones of DNA fromCurvularia lunata andBipolaris maydis as probes. Clear differences among species in both genera, resulting in different banding positions, were obtained with some probe-enzyme combinations. Intraspecific polymorphism in banding positions with these probe-enzyme combinations was slight. These analyses allow discrimination between the species. DNA fingerprinting with intrageneric probes is a potentially useful tool for species separation and identification inBipolaris andCurvularia when coupled with another characteristic such as conidial morphology.Curvularia aeria comb. nov. was proposed forCurvularia lunata var.aeria on the basis of differences in RFLP banding patterns and differences in conidial morphology.

Genetic analysis and characterization of Cochliobolus heterostrophus colour mutants

Twenty-three colour mutants of Cochliobolus heterostrophus were obtained by mutagenesis. Mutants at six loci were identified; alb1 (type-1 white), alb3 (type-2 white), sal1 (salmon), brn1 (brown), pgr1 (pale green), and scr1 (scarlet). Crossing experiments indicated that sal1 and pgr1 were in the same linkage group 5·8% apart, and that alb1, alb3 , and brn1 were closely linked. No linkage was observed between the three closely linked genetic markers ( alb1, alb3 , and brn1 ) and sal1 or pgr1. scr1 was independent of both of these linkage groups. Furthermore, no linkage was found between these loci and the mating type ( MAT1 ) locus. Accumulation of scytalone in the sal1 mutant indicated that the melanin of this fungus is formed from 1,8-dihydroxynaphthalene, derived in turn from pentaketide, and that the genetically deficient step in this mutant was at the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthesis pathway. The pgr1 mutant failed to form melanin from 1,8-dihydroxynaphthalene, which suggested that the deficiency of the pgr1 mutant is involved in oxidation of 1,8-dihydroxynaphthalene. The conversion of 1,3,8-trihydroxynaphthalene to vermelone may be blocked in the brn1 mutant. The defect of the alb1 mutant seemed to be involved in some process prior to scytalone formation. The alb3 mutant, which was not coloured by scytalone, was one of the melanin-deficient mutants, but its genetically blocked point was not characterized.

Molecular phylogeny and biogeography of the widely distributed Amanita species, A. muscaria and A. pantherina

The molecular phylogeny and biogeography of two widely distributed Amanita species, A. muscaria and A. pantherina, were studied based on specimens from diverse localities. Analyses of both a partial sequence of the ITS region of nuclear DNA and a partial sequence of the beta-tubulin gene were able to resolve specimens of each species. Analyses revealed a greater divergence of the beta-tubulin region than the ITS region. Based on molecular phylogeny of the combination of the ITS and beta-tubulin regions, A. muscaria could be separated into at least three groups (Eurasian, Eurasian subalpine, and North American), and A. pantherina could be separated into at least two groups (North American and Eurasian). We hypothesize that the speciation of A. muscaria occurred in Eurasia with subsequent migration to North America via land bridges. However, it is impossible to determine whether A. pantherina moved from Eurasia to North America or vice versa. For both A. muscaria and A. pantherina, the intracontinental relationships of both Eurasia and North America were closer than the relationships between eastern Asia and eastern North America.

Molecular phylogeny of Japanese Amanita species based on nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal DNA

The molecular phylogeny of 36 specimens of JapaneseAmanita species was studied based on nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The phylogenetic tree obtained supported the traditional classification systems of Bas (1969) and Singer (1986), which are based on morphological characters, in the division of the genusAmanita is divided into subgeneraAmanita andLepidella by the amyloidity of basidiospores. However, at section-level, we suggest that subgenusAmanita should be divided into three sections (Amanita, Vaginatae, andCaesareae). Our results also showed the necessity to modify the taxonomic treatments at section-level in the subgenusLepidella. It appears that the establishment ofA. muscaria andA. pantherina from a common ancestral species might be a very recent event, or these might be lower taxa of same species. As for three subspecies ofA. hemibapha and three varieties ofA. vaginata, it is necessary to grade up their taxonomical ranks from subspecies/variety to species. A new combination,A. javanica, is proposed forA. hemibapha subsp.javanica.

Genetic analysis of genes involved in melanin biosynthesis of Cochliobolus miyabeanus

Abstract Color mutants of Cochliobolus miyabeanus defective in melanin biosynthesis were isolated. Although the wild-type strain KU-13 formed dark green colonies, color mutants formed white, brown, and gray colonies or white colonies with red pigment secretion. From the white mutant which secreted red pigment, designated scy , a melanin precursor which restored melanization of albino mutants alm-1 was isolated and identified as scytalone. This indicated that scy mutant was defective in the conversion of scytalone to 1,3,8-trihydroxynaphthalene and that melanin of this fungus is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene. Albino mutants alm-1 were considered to be defective in pentaketide cyclization and brown mutants brm were considered to be defective in the conversion of 1,3,8-trihydroxynaphthalene to vermelone. Albino mutants alm-2 whose coloration was not restored by application of scytalone were also isolated. The alm-2 gene was believed to be a gene transactively regulating the pentaketide cyclization and conversion of scytalone. From crossing experiments among the color mutants, it was indicated that alm-1, alm-2 , and brm were linked and that scy segregates independently of these three mutant loci. Crossing of a methionine requiring mutant with alm and scy indicated that the three loci segregate independently of each other.

Induction of clovamide by jasmonic acid in red clover

The effect of jasmonic acid (JA) on the secondary metabolism of 5-day-old red clover seedlings was investigated. Induction of the formation of four compounds was found in roots after treatment with 50 microM JA for 48 h, while no induction was observed in the shoots. These compounds, whose formation was induced by JA addition, were isolated and identified as caffeoyl DOPA (clovamide), caffeoyltyrosine, p-coumaroyl DOPA and p-coumaroyltyrosine, by ion-spray MS and 1H NMR analyses, and by chemical synthesis. Among them, clovamide was the most abundant, while the other amides represented only a minor portion. Clovamide started to increase in amount 24-36 h after treatment and reached a maximum after 96 h (2.81 nmol/mg fr. wt.). The induction of their formation was observed even with 5 microM of JA, and the amount increased with concentrations up to 100 microM. Treatment with 1 mM CuCl2, which elicits accumulation of the phytoalexin maackiain in red clover, caused a decrease in clovamide amount.

The Pgr1 mutant of Cochliobolus heterostrophus lacks a p-diphenol oxidase involved in naphthalenediol melanin synthesis

1,8-Dihydroxynaphthalene (1,8-DHN) was isolated from Cochliobolus heterostrophus pale green mutant M13BL19 ( Alb1 + Brn1 + Sal1 + Pgr1-1 ) as the active substance which restored melanization of HE1AS73 ( Alb1-8 Brn1-2 Sal1-1 Pgr1 + ). HE1AS73 oxidized 1,8-DHN, 1-naphthol, N,N -dimethyl- p -phenylenediamine, tetramethylbenzidine, syringaldazine, and 3-(3,4′-dihydroxyphenyl)alanine, but did not oxidize p -cresol and l -tyrosine. None of these compounds was oxidized by HE5R13 ( Alb1-8 Brn1-2 Sal1-1 Pgr1-1 ). The syringaldazine oxidation activity of HE1AS73 was inhibited by diethyldithiocarbamate, cetyltrimethylammonium bromide, and dithiopyrimidine. These results indicate that the Pgr1 + strain contained p -diphenol oxidase which was absent from the Pgr1 − strain. We consider from these results that p -diphenol oxidase, in which Pgr1 is involved, plays a role in melanin synthesis of C. heterostrophus and 1,8-DHN is a natural substrate of this enzyme.