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Plants live in close association with large communities of microbes, some of which are foliar pathogens that invade tissues, primarily via stomata on the leaf surface. Stomata are considered part of an integral, innate immunity system capable of efficiently preventing pathogens from entering the host plant. Although Bacillus, a typical plant growth-promoting rhizobacterium, is known to induce stomatal closure, the substances participating in this closure and the mechanism involved in its regulation remain poorly understood. Here, we screened a mutant library and conducted site-specific mutagenesis experiments in order to identify such substances. We found that acetoin and 2,3-butanediol from B. amyloliquefaciens FZB42 induced stomatal closure in Arabidopsis thaliana and Nicotiana benthamiana. These two components could function either via root absorption or volatilization to restrict stomatal apertures, but root absorption was more efficient. Both substances invoked the salicylic acid and abscisic acid signaling pathways to close the stomata and stimulated accumulation of hydrogen peroxide and nitric oxide. The results present comprehensive evidence of how soil rhizobacteria may affect plant stomata, in a way that reinforces the evolved mutualism between the two groups of organisms, and provide potential alternative avenues of research towards reducing the incidence of disease in crops. [ABSTRACT FROM AUTHOR]