Disease Resistance Against a Broad-Host-Range Pathogen

The bacterial wilt pathogen Ralstonia solanacearum causes major agricultural losses on many crop hosts worldwide. Resistance breeding is the best way to control bacterial wilt disease, but the biological basis for bacterial wilt resistance is unknown. We found that R. solanacearum uses an AvrE-family, Type III-secreted effector called PopS to overcome plant defenses and cause disease on tomato. Orthologs of PopS are widely conserved across distinct classes of plant pathogenic bacteria and could provide novel, durable targets for resistance. Bacterial Wilt Disease The plant pathogenic bacterium Ralstonia solanacearum causes bacterial wilt disease of over 200 plant species in tropical and subtropical regions (6). This soilborne pathogen enters hosts through roots and colonizes the watertransporting xylem tissue, through which it spreads up into the plant stem (6). R. solanacearum infection reduces water transport, which eventually leads to plant wilting and death (Fig. 1). Fig. 1. Time-lapse video of bacterial wilt of tomato. This video shows bacterial wilt disease progress on wiltsusceptible tomato (cv. Bonny Best) caused by R. solanacearum strain UW551. To allow for a naturalistic infection, the soil of unwounded tomato plants was drenched with 50 mL of either a bacterial cell suspension (5 × 10 CFU/g soil) (left) or with water alone as a control (right). Plants were grown at 24°C 14 h light/19°C 10 h dark (day/night). Photos were captured every 10 sec for 26 days post inoculation and assembled with Time Lapse Assembler program. Only daytime images are displayed. vimeo.com/74855449. 8 25 November 2013 Plant Health Progress R. solanacearum forms a species complex, a heterogeneous group of xyleminfecting strains with variable host ranges, ecological traits, and levels of aggressiveness (6). The species complex is divided into four phylotypes that correlate with geographic origin (19). Genome sequencing of genetically and phenotypically diverse R. solanacearum strains has delineated the phylogenetic relationships within the complex. Genomic comparisons using average nucleotide identity (ANI) across entire genomes of over a dozen strains indicate that the species complex comprises three distinct proposed bacterial species: R. solanacearum (phylotype II); R. sequeirae (phylotype I and III); and R. syzygii (phylotype IV) (20). Growers suffer major losses worldwide to this broad host range pathogen, and in particular R. solanacearum limits tomato production in the southeastern United States. Because of a recent bacterial wilt epidemic in the eastern shore of Virginia, the third largest producer of fresh market tomatoes, R. solanacearum is currently considered Virginia’s most important tomato pest, causing up to 85% disease incidence in the field (Steven Rideout, personal communication). As with most soilborne bacterial diseases, bacterial wilt is difficult to control, and resistance breeding is the best method to mitigate crop losses to the disease. Bacterial Wilt Resistance Many plants have resistance (R) proteins that recognize microbial attack and limit infection by signaling a programmed cell death called the hypersensitive response (HR) (2). Gram-negative plant pathogenic bacteria, including R. solanacearum, secrete proteins called effectors into host cells through a molecular syringe called the Type III secretion system (T3SS) (1). Many Type III (T3)-secreted effectors are virulence determinants that disarm host defenses and/or alter plant physiology (1). Plant R-proteins often detect T3-secreted effectors and respond with an HR (2). There are few examples of R-proteinmediated resistance to R. solanacearum infection in agronomically important crops. For instance, some lines of tobacco (Nicotiana tabacum) recognize R. solanacearum T3-secreted effectors AvrA and PopP1, and the resulting plant defenses impede pathogen infection and bacterial wilt development (4,18,22). Recent work by Jeong and colleagues (12) demonstrated that a secreted but T3SS-independent aspartic protease, Rsa1, elicits HR and blocks R. solanacearum infection in pepper. However, no single gene resistance is available for important wilt-susceptible crops such as banana, potato, peanut, eggplant, and tomato. The quantitatively resistant tomato line Hawaii 7996 (H7996) is the most widely used source of resistance against bacterial wilt disease of tomato. H7996 has at least five quantitative trait loci (QTLs) that together confer resistance to most tropical R. solanacearum strains (24). Commercial deployment of H7996 resistance is difficult and slow because the basis of resistance to bacterial wilt is multigenic and not well understood. Our group demonstrated that when R. solanacearum infects tomato, it elicits expression of plant defense genes regulated by two major signaling pathways, the salicylic acid (SA) and ethylene pathways (15). Interestingly, bacterial extracellular polysaccharide, a major pathogenicity factor, is a specific elicitor of SA-mediated defense gene induction in H7996, but a wilt-susceptible tomato line did not recognize R. solanacearum extracellular polysaccharide (15). The specific mechanisms that trigger plant defenses and ultimately attenuate wilt progress are unknown not only for tomato, but for this pathogen’s other hosts as well. Conserved T3 Effector PopS to Overcome Resistance To infect, colonize host xylem tissue, and cause disease, R. solanacearum employs a suite of virulence factors that include the secreted effector proteins and extracellular polysaccharide mentioned above, as well as plant cell wall degrading enzymes, motility, and Type 4 pili (6). It was previously suggested that R. solanacearum only used its T3SS for early steps in host invasion (7,25), but recent evidence demonstrates that this pathogen also actively expresses its 25 November 2013 Plant Health Progress T3SS and effectors during stem colonization and early tomato wilt (11,16). This finding suggests that T3 secreted effectors have a role during later stages of disease. Most R. solanacearum strains possess over 70 putative effectors (17), but the biological activity of individual effectors during tomato infection remains undetermined. We found that during tomato wilt R. solanacearum expresses a gene encoding a highly conserved T3-secreted effector called PopS (locus tag Rsp1281 in strain GMI1000 and RRSL_03375 in strain UW551) (11) (phylotype II sequevar 1, formerly known as race 3 biovar 2). popS is part of the R. solanacearum core genome, which are the genes present in all sequenced R. solanacearum strains, including the fastidious Sumatra disease of clove pathogen, R. syzygii, and the banana Blood Disease Bacterium (8,20,21). The R. solanacearum core genome is proposed to encode the minimal traits necessary to be a bacterial wilt pathogen (21). Because of its wide conservation and its robust expression in planta, we characterized the role of PopS during tomato infection. We found that a popS mutant in strain UW551 was significantly delayed in virulence on wilt-susceptible tomato plants (cv. Bonny Best) but the popS mutant had a more dramatic virulence defect on quantitatively resistant H7996 (data not shown). PopS belongs to the widely distributed AvrE family of effectors, which are found in major Gammaproteobacterial plant pathogens such as Erwinia amylovora (DspE , fire blight of apple and pear), Pantoea stewartii subsp. stewartii (WtsE , Stewart’s wilt of corn), Pectobacterium carotovorum subsp. carotovoraum (DspE , bacterial soft rot), Pseudomonas syringae pv. tomato (AvrE and HopR , bacterial speck of tomato), and Xanthomonas campestris pv. campestris (HopR , black rot of cabbage) (Fig. 2) (5,9,13,14). The genomes of E. amylovora and P. stewartii subsp. stewartii encode few effectors, including a single AvrE-family effector. Strikingly, the AvrE-like effectors from these plant pathogenic enterics are major pathogenicity factors, meaning that these organisms are unable to cause disease without them (3,9). In contrast, Pseudomonas syringae pv. tomato has a large effector repertoire. Two of these, HopR and AvrE, belong to the AvrE effector family, but they do not contribute to virulence individually and their immune suppressing functions overlap with those of other P. syringae effectors (5,14). R. solanacearum PopS is not a pathogenicity factor like enteric AvrE orthologs, but it is a virulence factor because a popS mutant is significantly less virulent on tomato than its wild-type parent. Ea