Ralstonia solanacearum Needs Flp Pili for Virulence on Potato

dBurkholderia glumae PG1 is a soil-associated motile plant-pathogenic bacterium possessing a cell density-dependent regulation system called quorum sensing (QS). Its genome contains three genes, here designated bgaI1 to bgaI3, encoding distinct autoinducer-1 (AI-1) synthases, which are capable of synthesizing QS signaling molecules. Here, we report on the construction of B. glumae PG1 ⌬bgaI1, ⌬bgaI2, and ⌬bgaI3 mutants, their phenotypic characterization, and genome-wide transcriptome analysis using RNA sequencing (RNA-seq) technology. Knockout of each of these bgaI genes resulted in strongly decreased motility, reduced extracellular lipase activity, a reduced ability to cause plant tissue maceration, and decreased pathogenicity. RNA-seq analysis of all three B. glumae PG1 AI-1 synthase mutants performed in the transition from exponential to stationary growth phase revealed differential expression of a significant number of predicted genes. In comparison with the levels of gene expression by wild-type strain B. glumae PG1, 481 genes were differentially expressed in the ⌬bgaI1 mutant, 213 were differentially expressed in the ⌬bgaI2 mutant, and 367 were differentially expressed in the ⌬bgaI3 mutant. Interestingly, only a minor set of 78 genes was coregulated in all three mutants. The majority of the QS-regulated genes were linked to metabolic activities, and the most pronounced regulation was observed for genes involved in rhamnolipid and Flp pilus biosynthesis and the type VI secretion system and genes linked to a clustered regularly interspaced short palindromic repeat (CRISPR)-cas gene cluster. Q uorum sensing (QS) is a cell density-dependent gene regulation system in bacteria (1) in which the population density is sensed through the accumulation of bacterially produced signaling molecules called autoinducers (AIs). This cell-to-cell signaling process allows the microbial population to synchronize group behavior and alter its gene expression accordingly. QS is involved in a wide array of regulatory circuits, among which are pathogenicity, secretion of extracellular proteins, secondary metabolite production, and others (2). Key QS signaling molecules in many Gram-negative bacteria are N-acyl-homoserine lactones (AHLs) (3-5), synthesized mainly through LuxI homologs (EC 2.3.1.184) using S-adenosylmethionine (SAM), and an acyl-acyl carrier protein (acyl-ACP) from the fatty acid biosynthesis pathway (6). LuxR-type receptor/regulator proteins are involved in AHL signal perception. Together with LuxR, other proteins may be part of this regulatory circuit.The motile, rod-shaped Gram-negative soil bacterium Burkholderia glumae is considered to be a seed-borne pathogen that causes panicle blight of rice (7). B. glumae has also been reported to infect other plant species, like tomato, sunflower, and pepper (8, 9). Although it is not classified as a human pathogen, a single case of the isolation of B. glumae from a clinical sample was reported (10), indicating that at least some strains of this pathogen may be associated w...

Section: Global Pattern Of Qs-dependent Gene Expression In Bgpg1supporting

confidence: 61%

Section: Global Pattern Of Qs-dependent Gene Expression In Bgpg1mentioning

confidence: 99%

Genome-Wide RNA Sequencing Analysis of Quorum Sensing-Controlled Regulons in the Plant-Associated Burkholderia glumae PG1 Strain

Gao

Krysciak

Petersen

et al. 2015

“…Details about the characteristics of Tfpb in other systems, specifically, plant-associated bacteria, are only slowly emerging (19). The genome of the plant symbiont S. meliloti contains two gene clusters coding for Tfpb which belong to the Flp (fimbrial lowmolecular-weight protein) subfamily.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the importance of Tfp in plant-pathogen interactions is demonstrated in Xylella fastidiosa, where they help in the spread of the pathogen through grapevines (17). They also act as a virulence factor for Pseudomonas syringae in tobacco and Ralstonia solanacearum in potato plants (18,19). Despite the presence of Tfp genes in rhizobia, their role in bacterium-plant symbiosis is currently unknown.…”

mentioning

confidence: 99%

ExpR Coordinates the Expression of Symbiotically Important, Bundle-Forming Flp Pili with Quorum Sensing in Sinorhizobium meliloti

Zatakia

Nelson

Syed

et al. 2014

Type IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction of S. meliloti and its host, Medicago sativa, we deleted pilA1, which encodes the putative pilin subunit in the chromosomal flp-1 cluster and conducted competitive nodulation assays. The pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundleforming pili protruding from the polar and lateral region of S. meliloti wild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis of S. meliloti with its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve a coordinated expression of cellular processes during early stages of host interaction.

“…In addition, type IV pili (T4P) have also been shown to be involved in virulence of pathogenic bacteria, including P. aeruginosa, Neisseria meningitidis, and Ralstonia solanacearum (51)(52)(53). In this study, the PilZ proteins were found to regulate sliding motility, among which PXO_00049 acts as a positive regulator, whereas both PXO_02374 and PXO_02715 function as the negative ones.…”

Section: Fig 6 Subcellular Localization Of Pilz Domain Proteins In Pxo99mentioning

confidence: 81%

The Xanthomonas oryzae pv. oryzae PilZ Domain Proteins Function Differentially in Cyclic di-GMP Binding and Regulation of Virulence and Motility

Yang

Tian

Chen

et al. 2015

bThe PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R 141 and R 10 residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the fulllength open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae. Cyclic dimeric GMP (c-di-GMP) was originally identified as a positive allosteric regulator of cellulose synthase in Gluconacetobacter xylinus (1). Now, it has been recognized as an important intracellular signaling molecule implicated in the control of various bacterial properties, including motility, biofilm formation, adherence, and production of virulence factors (2, 3). The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs), which contain the GGDEF domain, and phosphodiesterases (PDEs), which harbor EAL or HD-GYP domains (4). Also, the downstream cellular functions regulated by c-di-GMP often depend on signal transduction by its receptors or effectors. A wide variety of c-di-GMP receptors have been discovered, such as PilZ domains (5), the transcription factors (6-8), proteins containing degenerate GGDEF or EAL domains (9-11), and the RNA processing polynucleotide phosphorylase (PNPase) and riboswitches (12-14). Therefore, c-di-GMP can exert a regulatory function at various levels of gene transcription, translation, and posttranslation.The PilZ domain is the first c-di-GMP binding module identified in G. xylinus and is named PilZ domain after being identified from Pseudomonas aerug...