Domain Structure of HrpE, the Hrp Pilus Subunit of Xanthomonas campestris pv. vesicatoria

Abstract

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and 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. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3⌬2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.Many gram-negative bacterial pathogens, including Xanthomonas campestris pv. vesicatoria, possess a unique protein transport system called the type III secretion (TTS) system which transfers so-called effector proteins directly into the host cell. In plant bacterial pathogens, genes encoding the TTS system are referred to as hypersensitive response and pathogenicity (hrp) genes because mutations in these genes abolish induction of the hypersensitive response in resistant host plants and pathogenicity in host plants (36).In X. campestris pv. vesicatoria, the hrp gene cluster is located in a 23-kb chromosomal region and is organized into six operons, designated hrpA to hrpF (6,17,18,24,25,48,58). Twenty-two genes are encoded within the hrp gene cluster, 11 of which are highly conserved among plant and animal pathogens and therefore have been renamed hrc (hrp conserved) genes (5, 56). Eight hrc gene products are associated with bacterial membranes and build up a transenvelope multiprotein complex (21). The remaining three proteins localize in the cytoplasm and are probably involved in energizing the transport process (20,44). The role of the non...

Positive Selection of the Hrp Pilin HrpE of the Plant Pathogen Xanthomonas

“…Notably, only the first four hydrophilic stretches of the HrpE sequence were positively selected, whereas the C-terminal hydrophilic stretch was not. These findings are in perfect agreement with the proposed domain structure of HrpE (29). In this previous work it was found that the N-terminal two-thirds of the protein are tolerant to pentapeptide insertions, whereas the C-terminal third is not.…”

“…Notably, for the C-terminal hydrophilic region an indication of positive selection is missing. This finding may be related to the proposed function of this domain in the polymerization process (29).…”

“…The very-N terminus of HrpE was shown to serve as a TTS signal (29). Despite 15 years of intense research, and even with the three-dimensional structures of the N termini of TTS substrates at hand, the nature of the TTS signal is still enigmatic (24).…”

“…Sequence alignment and secondary structure predictions of five hrpE sequences showed that the very-C terminus is entirely conserved and predominantly ␣-helical, whereas the N terminus is hypervariable and mostly devoid of regular secondary structure. Reporter protein fusions and pentapeptide insertions demonstrated that HrpE contains two functional domains, a surface-exposed domain, including the TTS signal, and the polymerization domain (29). The sequence hypervariability of the surface-exposed domain prompted us to analyze the evolutionary rate of 26 hrpE sequences, representing Xanthomonas strains belonging to 14 different pathovars.…”

“…The Hrp pilus is a filamentous structure that extends up to several micrometers from the bacterial cell surface and is mainly composed of a large number of identical pilin subunits, the 10-kDa HrpE protein, which is unique for the genus Xanthomonas (30). HrpE itself is a TTS substrate and harbors a secretion signal in the first 17 Nterminal amino acids (29). Sequence alignment and secondary structure predictions of five hrpE sequences showed that the very-C terminus is entirely conserved and predominantly ␣-helical, whereas the N terminus is hypervariable and mostly devoid of regular secondary structure.…”