Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance

Abstract

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...

Novel type of adenylyl cyclase participates in tabtoxinine-β-lactam-induced cell death and occurrence of wildfire disease inNicotiana benthamiana

“…Next, we analyzed the effect of NbAC silencing on cell death induction by MSX, specific inhibitor of glutamine synthetase that mimic the TβL. 15,16 Cell death was detected at 48 h after infiltration in control plants, whereas MSX-induced cell death and necrotic lesions were compromised in NbAC-silenced plants (Fig. 3A,B lower).…”

Distinct Phosphatase Requirements and GATA Factor Responses to Nitrogen Catabolite Repression and Rapamycin Treatment in Saccharomyces cerevisiae

“…GATA Factor-specific Responses to Methionine Sulfoximine Inhibition of Glutamine Synthetase-The final set of observations pointing to the distinct regulation of Gln3 and Gat1 localization derive from experiments with Msx (43,44,57). Treating cells with Msx results in rapid and complete nuclear Gln3-Myc 13 and Gln3-GFP localization (Ref.…”

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes

“…Although not considered previously in the literature, this is an expected result. Msx is, among other things, an effective glutamine synthetase inhibitor (21). Starving the cells for glutamine will cause the cessation of protein synthesis as the internal reserves of the amino acid are exhausted.…”