Concurrent Loss in Tn5Mutants ofPseudomonas syringaepv.syringaeof the Ability to Induce the Hypersensitive Response and Host Plasma Membrane K+/H+Exchange in Tobacco

Abstract

Stimulation of a plasmalemma K+ efflux/H+ influx exchange in tobacco [Nicotiana tabacum] by phytopathogenic bacteria has been shown to correspond with development of the hypersensitive response. To further test this relationship, mutants of Pseudomonas syringae pv. syringae were tested for loss of ability to induce the hypersensitive response or stimulate the K+/H+ exchange. Mutants were produced by using plasmid vector pGS9 to introduce transposon Tn5 into P. s. pv. syringae. Ability to induce the hypersensitive response was assayed by infiltrating mutants, at a concentration of 5 .times. 107 cfu/ml, into tobacco leaves. Stimulation of the K+/H+ exchange was detected by incubating suspension-cultured tobacco cells with mutants at a concentration of 108 cfu/ml for 18 hr, then testing for an increase in pH of the medium with a pH indicator. Colonies that failed to stimulate H+ influx were assayed quantitatively in a second assay that also confirmed the absence of K+ efflux from suspension-cultured tobacco cells. All mutants that failed to induce one response, either the hypersensitive response or the K+/H+ exchange response, also failed to induce the second response. Of 1,600 mutants screened, about 1.7% were auxotrophic. Six prototrophic mutants completely failed to induce either response, and three were able to only partially induce either response; all other prototrophs induced both responses. The results demonstrate a close relationship between bacterial induction of the K+/H+ exchange and the hypersensitive response.