Katsuyoshi Yoneyama

Trichothecene 3-O-Acetyltransferase Protects Both the Producing Organism and Transformed Yeast from Related Mycotoxins CLONING AND CHARACTERIZATION OF Tri101

Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms. The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system. The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism forFusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol). A gene responsible for the 3-O-acetylation reaction,Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed inSchizosaccharomyces pombe. Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101. This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms. The open reading frame ofTri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far. TRI101 from recombinant Escherichia coli catalyzesO-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner. By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F. graminearum. Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones. These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.

Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin

SummarySome plant pathogens produce toxins which cause disease in infected plants. One of the pathogenic toxins, tabtoxin, is produced by Pseudomonas syringae pv. tabaci, which causes wildfire of tobacco. A tabtoxin resistance gene (ttr) coding for an acetyltransferase isolated from Pseudomonas syringae pv. tabaci was fused to the 35S promoter of the cauliflower mosaic virus (CaMV) to construct a chimeric gene for introduction into tobacco cells by Agrobacterium-mediated transformation. The transgenic tobacco plants showed high specific-expression of the ttr gene and no chlorotic symptoms caused by tabtoxin treatment or with infection by Pseudomonas syringae pv. tabaci. These results demonstrate a successful approach to obtain disease-resistant plants by detoxification of the pathogenic toxins which play an important role in pathogenesis.

The mystery of the trichothecene 3-O-acetyltransferase gene: Analysis of the region around Tri101 and characterization of its homologue from Fusarium sporotrichioides

The trichothecene 3‐O‐acetyltransferase gene, Tri101, plays a pivotal role for the well‐being of the type B trichothecene producer Fusarium graminearum. We have analyzed the cosmids containing Tri101 and found that this resistance gene is not in the biosynthetic gene cluster reported so far. It was located between the UTP‐ammonia ligase gene and the phosphate permease gene which are not related to trichothecene biosynthesis. These two ‘house‐keeping’ genes were also linked in Fusarium species that do not produce trichothecenes. The result suggests that the isolated occurrence of Tri101 is attributed to horizontal gene transfer and not to the reciprocal translocation of the chromosome containing the gene cluster. Interestingly, 3‐O‐acetylation was not always a primary self‐defensive strategy for all the t‐type trichothecene producers; i.e. the type A trichothecene producer Fusarium sporotrichioides did not acetylate T‐2 toxin in vivo although the fungus possessed a functional 3‐O‐acetyltransferase gene. Thus Tri101 appears to be a defense option which the producers have independently acquired in addition to their original resistance mechanisms.

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

SummaryTransgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3′ end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R0 tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.

Cloning of the pathogenicity-related gene FPD1 in Fusarium oxysporum f. sp. lycopersici

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.

Assessment of Gibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.

Essential regulator gene toxR for toxoflavin biosynthesis of Burkholderia glumae

Burkholderia glumae (synonym: Pseudomonas glumae) is the causal agent of rice grain rot and seedling rot. This bacterium produces toxoflavin as a virulence factor for disease elicitation. Toxoflavin biosynthesis is completed by the transfer of methyl groups with catalytic action of a methyltransferase that is encoded by the toxA gene. In this study, we identified a 900-bp nucleotide sequence as a candidate gene to regulate the toxA gene. It was located upstream of the toxA gene. This novel regulatory element was named the toxR gene. When the toxR gene of B. glumae was disrupted by homologous recombination, one mutant (MY411) lost the ability to produce toxoflavin and to elicit the disease in rice seedlings. In addition, the expression of toxA mRNA was not detected by the reverse transcription-polymerase chain reaction, suggesting that the toxR gene is responsible for transcription of the toxA gene. The amino acid sequence deduced from the toxR gene was highly homologous to the LysR family transcriptional activators in some prokaryotes. Its amino-terminal had a helix-turn-helix DNA-binding motif to bind to a T-N11-A sequence motif of the toxA promoter. These results indicated that the toxR gene encoded the activator protein to promote transcription of the toxA gene and conceivably of downstream toxoflavin biosynthesis genes.

Epigenetic modification of rhizobial genome is essential for efficient nodulation

CcrM is one of the solitary bacterial DNA methyltransferases which does not have corresponding restriction enzymes. We established a stable ccrM-overexpressing mutant of Mesorhizobium loti, MlccrM-OX, and performed molecular and phenotypic characterization of this strain. In the M. loti MlccrM-OX infected plants, nodulation was apparently delayed at 7 days after inoculation (dai), however, the nodules that eventually formed on the MlccrM-OX roots showed nitrogen fixing ability by at least 21 dai. These results suggest that the initial morphogenic events were affected by ccrM-overexpression and that the correct pattern of DNA methylation of the bacterial genome is not essential for plant-microbe symbiosis, but are required for efficient nodulation.

Appearance of a new leaf rot disease on common ice plant

An unknown disease abruptly appeared on hydroponic cultures of common ice plant (Mesembryanthemum crystallinum) in the greenhouse, causing catastrophic damage. Although the symptoms of the plant were unlike typical Botrytis lesions on leaves and stems of other plants, fungi isolated from the necrotic lesions on the plant were similar to genus Botrytis in terms of conidial shape, colony color and nature. A representative isolate, Ice-2, caused similar symptoms on the host plants after inoculation with conidia, and the same fungus was isolated from the lesions. The conidia and conidiophores of Ice-2 were morphologically similar to those of B. cinerea but not to those of B.allii, B. fabae or B. squamosa. The tested Botrytis fungi grew at temperatures between 5 and 30°C. Ice-2 grew faster than the others at the lower end of the temperature range. Ice-2 was also more virulent than B. cinerea (Bay-1) in artificial inoculations, especially on common ice plant leaves. The glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) sequence of Ice-2 was determined and compared with those from four Botrytis species. The gene sequence of Ice-2 appeared to be identical to that of B. cinerea. In leaf tests on common ice plant and kidney bean, the diseases caused by Ice-2 and Bay-1 were controlled equally well by the primary Botrytis fungicides. Based on the results of the present studies, Ice-2 is thought to be Botrytis cinerea Person: Fries.

Social contributions of plant pathology with special focus on plant disease control

This article is an abstract of the Presidential Address presented at the 2004 Annual Meeting of the Phytopathological Society of Japan in Fukuoka Contributing to the betterment of society is a current concern in various academic fields in our country. In every area of academic science, including plant pathology, scientists have obviously made great contributions to solving problems and will continue to do so because science was born inevitably from our social needs. The problem is not science itself but scientists who do not correctly understand the social purpose of their science. Such responsibility of the scientist has now arisen as a social concern. Since the 1960s, plant pathology has progressed remarkably with the development of molecular biology and molecular genetics, and plant diseases have become understood as molecular or genetic phenomena. Molecular plant pathology has also promoted the development of other scientific and agricultural fields, such as microbiology, plant science, biotechnology, and crop breeding. Thereby, plant pathology came into the limelight as an attractive career in the life sciences. At the same time, however, plant pathologists generally have lost interest in the central concerns of plant pathology, such as diagnosis, microbial identification, and the control of plant diseases. This tendency, extending from institute to university, has caused a decline in the ability of plant pathologists, especially young plant pathologists, to diagnose plant diseases. This phenomenon has occurred not just in our country but in all advanced nations. Recognizing this deficiency, the International Phytopathological Society has noted the critical need for education in these basic areas of plant pathology in every country. In response, our society this year started an educational program on disease diagnosis for graduate students and young researchers. We should never forget that plant pathology is not for the welfare of plant pathologists but for the welfare of plants and that a principal aim of plant pathology is to keep plants healthy.