A novel luxR homolog, termed XagR, in Xanthomonas axonopodis pv. glycines, the cause of soybean pustule, controls expression of pip, yapH, and at least 77 other genes. Although XagR and Pip are required for full virulence of X. axonopodis pv. glycines to soybean, constitutive overproduction of XagR suppresses infection. The xagR-dependent induction of pip occurs in planta only 2 days or more after inoculation. Although the transcription of xagR appears constitutive, XagR accumulates only in cells that have colonized soybean plants for more than 2 days suggesting that some components produced during the infection process mediate post-transcriptional control, likely by protecting XagR from proteolytic degradation. XagR modulates the adhesiveness of the pathogen during the infection process by suppressing the adhesin YapH. Although yapH mutants incite more infections of soybean leaves than the wild-type strain when topically applied under dry conditions, the mutant causes fewer infections when leaves are subject to simulated rain events after inoculation. Likewise, yapH mutants and cells in which XagR was overexpressed exhibited much more egress from infected leaves than the wild-type strain. Thus, XagR differentially modulates expression of a variety of genes during the infection process in response to feedback from plant molecules elaborated during infection to coordinate processes such as invasion, infection, and cell egress needed to complete the disease cycle.

Summary The stringent response, mediated by second messenger (p)ppGpp, results in swift and massive transcriptional reprogramming under nutrient limited conditions. In this study, the role of (p)ppGpp on virulence of P. syringae pv. syringae B728a (PssB728a) was investigated. The virulence of the relA/spoT (ppGpp0) double mutant was completely impaired on bean, and bacterial growth was significantly reduced, suggesting that (p)ppGpp is required for full virulence of P. syringae. Expression of T3SS and other virulence genes was reduced in ppGpp0 mutants. In addition, ppGpp deficiency resulted in loss of swarming motility, reduction of pyoverdine production, increased sensitivity to oxidative stress and antibiotic tolerance, as well as reduced ability to utilize γ-amino butyric acid. Increased levels of ppGpp resulted in reduced cell size of PssB728a when grown in a minimal medium and on plant surfaces, while most ppGpp0 mutant cells were not viable on plant surfaces 24 h after spray inoculation, suggesting that ppGpp-mediated stringent response temporarily limits cell growth, and might control cell survival on plants by limiting their growth. These results demonstrated that ppGpp-mediated stringent response plays a central role in P. syringae virulence and survival, and indicated that ppGpp serves as a global signal for regulating various virulence traits in PssB728a.

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp 0 ) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp 0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp 0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and ␣-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.E rwinia amylovora causes a devastating fire blight disease of apples and pears, which results in severe economic losses to growers around the world (1, 2). E. amylovora is closely related to members of the Enterobacteriaceae family, including many important human pathogens, such as Escherichia coli, Yersinia pestis, and Salmonella enterica (3). Studies have revealed that the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a major pathogenicity factor in E. amylovora (4-7). The hrp T3SS genes are carried on a pathogenicity island (8), and the alternative sigma factor HrpL, a member of the ECF subfamily of sigma factors, serves as the master regulator to control the expression of the structural and effector genes by binding to a consensus sequence known as the hrp box (9-13)...

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Pseudomonas syringae pv. tomato DC3000 (DC3000). In this study, the role of the second messenger (p)ppGpp on virulence and survival of DC3000 was investigated. Results have demonstrated that (p)ppGpp-deficient mutant (ppGpp(0)) of DC3000 exhibited lower levels of expression of the T3SS and genes of other virulence traits, such as coronatine toxin. The ppGpp(0) mutant of DC3000 was greatly impaired in causing disease and in growth in planta. Furthermore, (p)ppGpp was required for swarming motility, pyoverdine production, the oxidative stress response, as well as γ-amino butyric acid utilization. Screening of amino acids, major signals in activation of ppGpp biosynthesis, revealed that promoter activities of the avrPto gene could be either activated or suppressed by various amino acids in a ppGpp-dependent or -independent manner. Moreover, the ppGpp(0) mutant exhibited increased cell size and decreased survival on plant surfaces. Altogether, these findings indicate that ppGpp acts as an internal signal that regulates the T3SS as well as other virulence factors in pseudomonads and suggest that bacterial pathogens utilize intracellular messengers to sense environmental and nutritional signals for rapid, precise, and reversible control of their pathogenesis and survival.

To better understand the behavior of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean within its host, its global transcriptome within soybean leaves was compared with that in a minimal medium in vitro, using deep sequencing of mRNA. Of 5,062 genes predicted from a draft genome of X. axonopodis pv. glycines, 534 were up-regulated in the plant, while 289 were down-regulated. Genes encoding YapH, a cell-surface adhesin, as well as several others encoding cell-surface proteins, were down-regulated in soybean. Many genes encoding the type III secretion system and effector proteins, cell wall-degrading enzymes and phosphate transporter proteins were strongly expressed at early stages of infection. Several genes encoding RND multidrug efflux pumps were induced in planta and by isoflavonoids in vitro and were required for full virulence of X. axonopodis pv. glycines, as well as resistance to soybean phytoalexins. Genes encoding consumption of malonate, a compound abundant in soybean, were induced in planta and by malonate in vitro. Disruption of the malonate decarboxylase operon blocked growth in minimal media with malonate as the sole carbon source but did not significantly alter growth in soybean, apparently because genes for sucrose and fructose uptake were also induced in planta. Many genes involved in phosphate metabolism and uptake were induced in planta. While disruption of genes encoding high-affinity phosphate transport did not alter growth in media varying in phosphate concentration, the mutants were severely attenuated for growth in soybean. This global transcriptional profiling has provided insight into both the intercellular environment of this soybean pathogen and traits used by X. axonopodis pv. glycines to promote disease.