A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

Abstract

SummaryGacS/GacA is a conserved two‐component system that functions as a master regulator of virulence‐associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA‐regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)‐encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain‐to‐strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re‐evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface‐inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.

Re-evaluation of a Tn5::gacA mutant of Pseudomonas syringae pv. tomato DC3000 uncovers roles for uvrC and anmK in promoting virulence

“…AC811 is unable to grow to high levels or cause disease to Arabidopsis [16–17]. To determine whether AC811 has a general growth defect, we cultured DC3000, AC811 and a DC3000 Δ gacA deletion strain in King’s B (KB) broth at 28˚C, a temperature frequently used for overnight culturing of P .…”

“…Decreased virulence of AC811 in the apoplast of Arabidopsis leaves is not due to loss of gacA [17]. We hypothesized that another mutation may be responsible for the decreased virulence of AC811.…”

“…Predictive models were developed primarily on the basis of characterizations of a single mutant strain AC811 that contains a Tn 5 transposon insertion within the gacA open reading frame [16]. In a recent study, we re-examined phenotypes of AC811 and discovered that, contrary to previous reports, this mutant strain has increased, rather than decreased, expression of T3SS genes during infection of Arabidopsis [17]. Furthermore, we demonstrated that decreased growth of AC811 within Arabidopsis leaves was not due to loss of GacA function.…”

Re-evaluation of a Tn5::gacAmutant ofPseudomonas syringaepv.tomatoDC3000 uncovers roles foruvrCandanmKin promoting virulence

“…AC811 is unable to grow to high levels or cause disease to Arabidopsis [16-17]. To determine whether AC811 has a general growth defect, we cultured DC3000, AC811 and a DC3000 Δ gacA deletion strain in King’s B (KB) broth at 28°C, a temperature frequently used for overnight culturing of P. syringae .…”

“…The previously described Δ gacA ::pK18 mobsacB suicide vector [17] was conjugated into DC3000 by tri-parental mating using an E. coli helper strain carrying pRK600 [43]. Merodiploid colonies were passaged 1-3 days in KB broth with 50 μg/mL rifampicin prior to counter-selection on KB agar with 15% sucrose.…”