A stress-induced mRNA was identified in the phytopathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Treatment of the fungus with ethanol resulted in the induction of a major mRNA species encoding a protein of approximate Mr 37,000. A full-length cDNA clone of the induced message was obtained. RNA blot analysis indicated that the mRNA was induced by various other stresses, including treatment with copper(II) chloride and heat (37 degrees C). However, it was not greatly induced by treatment with phaseollinisoflavan, an antifungal isoflavonoid produced by Phaseolus vulgaris (French bean). In contrast, phaseollinisoflavan induced the homologous mRNA in the related bean pathogen Fusarium solani f. sp. phaseoli. A genomic clone of the F. solani f. sp. phaseoli gene was obtained, and both this and the cDNA clone from F. oxysporum f. sp. cucumerinum were sequenced. The latter indicated an open reading frame of 320 codons encoding a 34,556-dalton polypeptide. The corresponding reading frame in F. solani f. sp. phaseoli was 324 codons, 89% identical to the F. oxysporum f. sp. cucumerium sequence, and was interrupted by a short intron. The gene was designated sti35 (stress-inducible mRNA). Although computer homology searches were negative, the cloned gene was observed to cross-hybridize to DNAs of other filamentous fungi, Saccharomyces cerevisiae, and soybean. Thus, sti35 appears to be a common gene among a variety of eucaryotes.

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Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.Wildfire is an infectious leafspot disease of tobacco. The chlorotic leaf symptom that characterizes the disease is caused by tabtoxin (32), a phytotoxic dipeptide produced by Pseudomonas tabaci. Tabtoxin is a novel ,-lactamcontaining amino acid, 2-amino-4-(3-hydroxy-2-oxoazacyclobutan-3-yl)butanoic acid, linked by a peptide bond to the amino group of either serine or threonine (32). Tabtoxin inhibits glutamine synthetase activity in tobacco in vivo, causing accumulation of ammonium (8,30,34). Inhibition of glutamine synthetase has at least two effects, either one of which could cause cell death. First, the enzyme is probably the only major route for glutamine synthesis in plants, and second, the glutamine synthetase-glutamate synthase pathway is the only efficient way to detoxify the ammonia released by nitrate reduction, amino acid degradation, or photorespiration in most plants (17).Inhibition of glutamine synthetase by tabtoxin can be prevented by glutamine (30), which suggests that glutamine synthetase is the only cellular target in tobacco. Methionine sulfoximine (MSO), known to be a potent and highly specific inhibitor of glutamine synthetase (22), causes chlorotic lesions in tobacco leaves, and these effects are also prevented by glutamine (30). Because it appears to mimic the effects of tabtoxin and because it is commercially available, whereas tabtoxin is not, MSO has been used as a tool for studying wildfire disease in tobacco. Carlson (3) isolated MSOresistant tobacco cells from callus cultures and showed that plants derived from them were resistant to the chlorotic affects of both MSO and P. tabaci. In some lines the MSO-resistant trait behaved as a single dominant locus. The molecular mechan...

A vector was constructed for transformation of the plant pathogenic fungus Fusarium solani. The promoter 35Sp, from cauliflower mosaic virus, was fused to the bacterial gene APH(3')II, which confers resistance to the aminoglycoside antibiotic G418. Two transformation procedures were developed: one using isolated fungal protoplasts, the other using germinated fungal spores. A transformation frequency of 3.3 G418-resistant colonies were obtained per microgram DNA. Of 14 colonies analyzed, 12 had vector sequences integrated into their high molecular weight DNA, and 2 were untransformed. Integration was sometimes accompanied by rearrangements of both the vector and flanking fungal DNAs. Primer-extension analysis of the mRNA from one transformant revealed two putative transcription initiation sites in the chimeric APH(3')II gene. Both sites differed from the normal initiation site in plants. This vector will be useful in transformation systems in which integration by non-homologous recombination is desired.