Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum

Abstract

Quorum sensing (QS) in vitro controls production of plant cell wall degrading enzymes (PCWDEs) and other virulence factors in the soft rotting enterobacterial plant pathogen Pectobacterium atrosepticum (Pba). Here, we demonstrate the genome-wide regulatory role of QS in vivo during the Pba-potato interaction, using a Pba-specific microarray. We show that 26% of the Pba genome exhibited differential transcription in a QS (expI-) mutant, compared to the wild-type, suggesting that QS may make a greater contribution to pathogenesis than previously thought. We identify novel components of the QS regulon, including the Type I and II secretion systems, which are involved in the secretion of PCWDEs; a novel Type VI secretion system (T6SS) and its predicted substrates Hcp and VgrG; more than 70 known or putative regulators, some of which have been demonstrated to control pathogenesis and, remarkably, the Type III secretion system and associated effector proteins, and coronafacoyl-amide conjugates, both of which play roles in the manipulation of plant defences. We show that the T6SS and a novel potential regulator, VirS, are required for full virulence in Pba, and propose a model placing QS at the apex of a regulatory hierarchy controlling the later stages of disease progression in Pba. Our findings indicate that QS is a master regulator of phytopathogenesis, controlling multiple other regulators that, in turn, co-ordinately regulate genes associated with manipulation of host defences in concert with the destructive arsenal of PCWDEs that manifest the soft rot disease phenotype.

Figure 1. OHHL is Required for Virulence in Potato Stems and Peaks at 16 hpi in P. atrosepticum-infected Potato Tubers.

(A) Lesion development on potato stems following inoculation of the wild type Pba1043 and expI mutant strains. Filled bars = potato cv Desiree control; open bars = OHHL-producing transgenic Desiree (YI5A) . (B) Cell density and OHHL production (in light units) of wild type P. atrosepticum and expI mutant strains in potato tubers over 120 h. Wild type cell density (square); expI mutant cell density (diamond); wild type OHHL production (circle); mutant OHHL production (triangle). Bars show mean +/− standard error of the mean.

Figure 2. OHHL Complementation of Selected Quorum Sensing-regulated Genes.

RT-PCR analysis of selected genes after 18h growth of wild type P. atrosepticum in Pel Minimal Medium (PMM), and growth of the expI mutant in PMM with and without the addition of OHHL (5 µM final concentration). vgrG = ECA2867. Bars show mean +/− standard deviation.

Figure 3. Expression of outD-gusA in vitro is QS-dependent.

β-glucuronidase activity from an outD-gusA reporter fusion was measured in a wild type P. atrosepticum background (wild type, squares), in an expI mutant (expI, triangles) and in an expI mutant with the addition of exogenous 1 µg/ml OHHL (expI+OHHL, circles) throughout growth in PMB. β-glucuronidase activity (solid symbols) is expressed as A405/min/ml/OD600 and growth was measured as OD600 (open symbols). Bars show mean +/− standard error of the mean.

Figure 4. The Regulator virS and Components of the Type VI Secretion System are Involved in Virulence.

Virulence assays in potato stems (A) and potato tubers (B), following inoculation of wild type P. atrosepticum and mutants affected in virS and Type VI secretion (ECA3444 and ECA3438). Complementation of tuber rotting phenotype using plasmids pGEM-T (ECA3444 and ECA3438) and pQE80-L (virS) (B). “C” indicates complemented. Bars show mean +/- standard error of the mean.

Figure 5. Schematic Model of the Hierarchical Relationships Between ExpI, Other Regulators and Virulence Factors in Pectobacterium.

OHHL is believed to act via antagonism of the LuxR-family AHL-responsive transcriptional regulator, VirR, in turn activating or repressing numerous other regulators that control virulence factor gene expression. A second LuxR-type repressor, ExpR, may also contribute to OHHL regulation of downstream genes. This model represents an integrated summary of regulatory data derived from the study of multiple Pectobacterium strains, and is unlikely to apply in every aspect to all such strains. Adapted from von Bodman et al., using additional information from Barnard and Salmond , and this study. Solid arrowheads indicate activation and bars indicate repression. Dotted lines indicate uncertain and/or strain dependent effects.