The Type III-Dependent Hrp Pilus Is Required for Productive Interaction of Xanthomonas campestris pv. vesicatoria with Pepper Host Plants

Abstract

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Pathogenic bacteria exploit different strategies to successfully colonize their eukaryotic hosts. One of the key bacterial pathogenicity mechanisms is the translocation of proteins into eukaryotic host cells by a type III secretion (TTS) system consisting of a trans-envelope multiprotein complex. Components of TTS systems are generally encoded by gene clusters which often reside in pathogenicity islands (25). Several TTS systems have been studied, but our knowledge on the mechanism of substrate recognition and translocation is still very limited. The injectisome of Yersinia is the prototype example of a TTS system (13). There are three hallmarks of type III protein secretion. First, upon secretion, there is no processing of the protein substrate (42). Second, targeting to the TTS system involves sequence information at the N terminus of the protein and/or the corresponding region of the mRNA (14).Third, secretion across the bacterial cell envelope appears to occur in one step without a periplasmic intermediate (11).Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease in pepper and tomato (8). The X. campestris pv. vesicatoria TTS system is encoded by a 23-kb chromosomal hrp (hypersensitive response and pathogenicity) gene cluster which contains six operons, hrpA to hrpF (Fig. 1) (5, 20, 21, 30, 51; U. Bonas, unpublished data). Loss of hrp gene function results in a pleiotropic phenotype: hrp mutants are unable to grow in the plant, no longer cause disease symptoms, and fail to induce the hypersensitive reaction in resistant host and nonhost plants (5). The hypersensitive reaction is a rapid, local, programmed cell death that is induced upon recognition of the pathogen and is concomitant with the inhibition of pathogen growth within the infected plant tissue (35).hrp gene expression is induced in planta (55) and is controlled by the regulatory genes hrpG and hrpX, which are located outside the hrp gene cluster. The HrpG protein belongs to the OmpR family of two-component regulatory systems (64) and controls the expression of a large gene regulon including hrpX. The AraC-type transcriptional activator HrpX regulates the expression of the operons hrpB t...

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…HrpB2 is essential for pilus formation and interacts with HpaC and the C-terminal domain of the YscU/ FlhB homolog HrcU (HrcU C ), which also provides a binding site for HpaC (330,332,496,591). Experimental evidence suggests that the HrcU C -HrpB2 interaction is required for the efficient secretion of HrpB2 prior to the substrate specificity switch, which is in agreement with the predicted role of HrcU C as a substrate acceptor site (330).…”

Death Don't Have No Mercy and Neither Does Calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and Innate Immunity

“…Quantified evaluations of HR development and concomitant tissue collapse using a standard HR scoring system (see Methods) from the same experiment are shown in Figure 1C. Ethanol bleaching has been used as a convenient assay to provide enhanced visualization of HR development in leaves responding to an avirulent pathogen (Schornack et al, 2004;Weber et al, 2005). HR development and tissue collapse in leaves of dnd1 plants pretreated with SNP and inoculated with Pss were visualized in another experiment (with a different set of plants) after ethanol bleaching ( Figure 1E).…”

Innate Immunity Signaling: Cytosolic Ca2+ Elevation Is Linked to Downstream Nitric Oxide Generation through the Action of Calmodulin or a Calmodulin-Like Protein

“…Darkened areas of the bleached leaves corresponding to pathogeninduced tissue necrosis (Schornack et al, 2004;Weber et al, 2005;Ali et al, 2007) were used to evaluate HR development in response to pathogen infection. Alternatively, HR in pathogen-inoculated leaves was evaluated as ion leakage associated with PCD (Rate and Greenberg, 2001;Devadas and Raina, 2002;Torres et al, 2002).…”