Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants

Abstract

HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and sitedirected mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were In many interactions between gram-negative plant-pathogenic bacteria and plants, hrp (for hypersensitive reaction and pathogenicity) genes are required for pathogenicity in the host plant and induction of the hypersensitive response (HR) in nonhost plants (17). Regions that contain a cluster of hrp genes and other components required for pathogenicity are designated pathogenicity islands (PAIs) (3, 15). Most hrp genes that encode components of the type III protein secretion system mediate the translocation of effector proteins, such as Avr (avirulence) proteins, across the bacterial membrane and the walls and plasma membranes of plant cells (10).HR is a highly localized plant cell death that occurs when nonhost plants or resistant cultivars of host plants are infiltrated with the plant pathogen or HR elicitor molecules, such as Avr proteins and harpins. HR is thought be a resistance reaction of plants to microbial pathogens (11). Harpins are a group of HR elicitors that are secreted by the type III secretion pathway and elicit HR when infiltrated into the apoplast of leaves of nonhost plants. Unlike Avr proteins, which must be delivered inside the cell to exert their functions, harpins can elicit HR when delivered to the intercellular space of plant cells (10). Since the first known harpin, HrpN, was identified from Erwinia amylovora, many harpins have been reported from Pseudomonas, Ralstonia, and Xanthomonas species (4,8,12,14,15,27). Harpins are glycine-rich, heat stable, and lack cysteine, but the biochemical mechanisms of HR elicitation in nonhost plants are unclear. One reason for this is that the amino acid sequences of harpins do not share significant homology with other known proteins or among themselves.The mode of action of harpins is still controversial. HrpZ of Pseudomonas syringae pv. syringae associates with the walls rather than the membranes of plant cells, and the protein elicits no response from protoplasts, which lack walls (13). However, HrpZ of P. syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore (16). The N-terminal 109 amino acids and the C-terminal 216 amino acids of HrpZ are able to elicit HR to a level similar to full-length HrpZ (2). Kim et al. and Charkowski et al. showed that the HrpW harpins of E. amylovora and P. syringae pv. tomato are composed of two domains-the N-terminal harpin domain and C-terminal Pel (pectate lyase) domain-and proposed that HrpW acts in the cell wall (8,14).We previously published the first report of a harpin from Xanthomonas species, HpaG (15). At 13.4 kDa, HpaG is smaller than other known harpins (15). Four additional...

Within-Species Flagellin Polymorphism inXanthomonas campestrispvcampestrisand Its Impact on Elicitation ofArabidopsis FLAGELLIN SENSING2–Dependent Defenses

“…This finding regarding flagellin does not imply that Xcc bacteria entirely escape detection by plant basal defense systems. For example, lipopolysaccharides, harpins, and cold shock proteins of X. campestris have been implicated as general elicitors of plant defense responses (Newman et al, 1995;Meyer et al, 2001;Felix and Boller, 2003;Kim et al, 2004;Silipo et al, 2005). Our results reveal that flagellin detection systems are apparently circumvented by Xcc bacteria, but plant-microbe interactions involve multiple layers of defense and counterdefense, and other elicitors of basal defenses remain that may limit plant colonization by Xcc.…”

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum , Are Translocators Belonging to the HrpF/NopX Family

“…glycines and Xanthomonas oryzae pv. oryzae (40,68), has been suggested to be a component of the type III translocon (54). Indeed, a xopA mutant is reduced in pathogenicity without affecting type III secretion in vitro.…”

Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

“…Similarly, the single-domain harpin and proposed translocator, XopA, of X. campestris pv. vesicatoria is truncated at a conserved alpha helix essential for exogenous HR elicitation by its homologs (35). The use of a variable consortium of translocators may benefit phytopathogens by keeping the concentration of individual translocators below the recognition thresholds of host surveillance systems and by enabling individual translocators that have come under host surveillance to be lost from the consortium without loss of overall translocon function.…”

“…Harpin-like proteins have been identified in all T3SS-utilizing phytopathogens (1,4,13,28,34,35,70). Although these proteins may have little homology, they share several characteristics, such as a low isoelectric point, enrichment for glycine, and a lack of cysteine residues.…”