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© 2013 Plant Management Network.
Accepted for publication 25 September 2013. Published 25 November 2013.


Disease Resistance Against a Broad-Host-Range Pathogen


Jonathan M. Jacobs, UMR 186 Résistance des Plantes aux Bioagresseurs, Institut de Recherche pour le Développement, Montpellier, France (formerly, University of Wisconsin-Madison); and Caitilyn Allen, Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706


Corresponding author: Caitilyn Allen. cza@plantpath.wisc.edu


Jacobs, J. M., and Allen, C. 2013. Disease resistance against a broad-host-range pathogen. Online. Plant Health Progress doi:10.1094/PHP-2013-1125-03-RS.


Abstract

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 water-transporting 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 wilt-susceptible 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 × 108 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.

 

R. solanacearum forms a species complex, a heterogeneous group of xylem-infecting 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-protein-mediated 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 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 (DspEEa, fire blight of apple and pear), Pantoea stewartii subsp. stewartii (WtsEPss, Stewart’s wilt of corn), Pectobacterium carotovorum subsp. carotovoraum (DspEPcc, bacterial soft rot), Pseudomonas syringae pv. tomato (AvrEPst and HopRPst, bacterial speck of tomato), and Xanthomonas campestris pv. campestris (HopRXcc, 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.


 

Fig. 2. Pathogenic distribution of PopS and the AvrE/HopR/DspE-family of effectors. Phylogenetic tree of PopS orthologs from R. solanacearum (Rs) UW551 (PopS, EAP73412.1), R. solanacearum GMI1000 (PopS, NP_522840.1) R. solanacearum Molk2 (PopS, WP_003277099.1), P. carotovorum subsp. carotovorum (Pcc) B100 (DspE, AFR03665) E. amylovora (Ea) (DspE, AAC04850), X. campestris pv. campestris (Xcc) B100 (HopR, YP_001904661.1), P. syringae pv. tomato (Pst) DC3000 (HopR1, NP_790722.1; AvrE, AAF71499.1). The tree was created with MEGA5 (23). Amino acid sequences were aligned with MUSCLE and used to create a Maximum-likelihood tree (bootstrap value, 200) (23). Images courtesy of C. Allen, A. Sanchez-Perez, P. S. McManus, A. M. Gevens, B. D. Hudelson and the UW-Madison Plant Disease Diagnostic Clinic.

 

AvrEPst and DspEEa are known to suppress salicylic acid-mediated defenses (5). We analyzed defense gene expression in tomato plants infected with either wild-type or popS mutant strains of R. solanacearum and found that PopS also suppresses SA-mediated defenses (data not shown). This suggests that suppression of SA-mediated plant defenses may be a selectively important trait that has been conserved among AvrE family effectors in species descended from a common ancestral plant-associated bacterium. Wilt-resistant H7996 tomato displays a stronger and earlier SA-dependent defense response to R. solanacearum infection than susceptible Bonny Best (15). We hypothesize that PopS helps R. solanacearum overcome SA-dependent resistance in H7996.

The AvrE effector family likely has ancient origins in plant pathogenesis because of its broad conservation across phylogenetically distant classes of plant pathogenic bacteria. Proteins in this family have likely adapted to each individual species and evolved specific roles in each pathosystem. For example, the Gammaproteobacterial plant pathogens with AvrE-like effectors all cause some form of necrosis on their plant hosts (Fig. 2). Interestingly, DspE causes cell death when transiently expressed in Nicotiana benthamiana leaves (10). In contrast, R. solanacearum is a wilt pathogen that does not cause necrosis. It will be interesting to determine if and how this family of effectors differs in function in the Betaproteobacterium R. solanacearum compared to in the many Gammaproteobacterial plant pathogens that carry AvrE orthologs.


Concluding Remarks

R. solanacearum has an unusually broad host range and it somehow evades specific recognition by most host plant species. The biological basis of this broad host range, which contributes significantly to this pathogen’s destructiveness, is not understood. A proximal explanation is that although R. solanacearum produces a large number of effectors, for unknown reasons very few of these are recognized by plant R-proteins. PopS is among R. solanacearum’s "invisible" effectors. Interestingly, no R-proteins are known to recognize any AvrE-family members. PopS and its many orthologs have a long history of intimate interactions with plants, presumably under strong selection pressure favoring disease resistance. The fact that plants appear not to have evolved the ability to recognize and resist these ancient conserved effectors suggests that their mode of action is quite surreptitious or that their plant targets are essential and highly conserved. Functional studies are needed to identify the plant target(s) of AvrE family effectors. Understanding their mode(s) of action is likely to be broadly important. For example, synthetic plant receptors engineered to recognize conserved elements of AvrE effectors could offer a general mechanism of resistance against many diverse bacterial plant pathogens.


Acknowledgments

The authors gratefully acknowledge the APS Foundation for supporting travel to the American Phytopathological Society Annual Meeting 2012 in Providence, RI, and to present this research in the 12th APS I. E. Melhus Graduate Symposium. This research was supported by Storkan-Hanes-McCaslin Foundation Award to J.M.J., by USDA-CSREES Plant Biosecurity grant 2006-04560 and by the University of Wisconsin-Madison College of Agricultural and Life Sciences.


Literature Cited

1. Alfano, J. R., and Collmer, A. 2004. Type III secretion system effector proteins: Double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42:385-414.

2. Bent, A., and Mackey, D. 2007. Elicitors, effectors, and R genes: The new paradigm and a lifetime supply of questions. Annu. Rev. Phytopathol. 45:399-436 doi:10.1146annurev.phyto.45.062806.094427.

3. Bogdanove, A. J., Kim, J. F., Wei, Z., Kolchinsky, P., Charkowski, A. O., Conlin, A. K., Collmer, A., and Beer, S. V. 1998. Homology and functional similarity of an hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato. Proc. Natl. Acad. Sci. USA 95:1325-1330.

4. Carney, B. F., and Denny, T. P. 1990. A cloned avirulence gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level. J. Bacteriol. 172:4836-4843.

5. DebRoy, S., Thilmony, R., Kwack, Y. B., Nomura, K., and He, S. Y. 2004. A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants. Proc. Natl. Acad. Sci. USA 101:9927-9932 doi:10.1073/pnas.0401601101.

6. Denny, T. 2006. Plant pathogenic Ralstonia species. In: Plant-Associated Bacteria. S. S. Gnanamanickam, ed. Springer Press, New York.

7. Genin, S., Brito, B., Denny, T. P., and Boucher, C. 2005. Control of the Ralstonia solanacearum Type III secretion system (Hrp) genes by the global virulence regulator PhcA. FEBS Lett. 579:2077-2081.

8. Guidot, A., Prior, P., Schoenfeld, J., Carrere, S., Genin, S., and Boucher, C. 2007. Genomic structure and phylogeny of the plant pathogen Ralstonia solanacearum inferred from gene distribution analysis. J. Bacteriol. 189:377-387.

9. Ham, J. H., Majerczak, D. R., Nomura, K., Mecey, C., Uribe, F., He, S. Y., Mackey, D., and Coplin, D. L. 2009. Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs. Mol. Plant Microbe Interact. 22:703-712 doi:10.1094/MPMI-22-6-0703.

10. Hogan, C. S., Mole, B. M., Grant, S. R., Willis, D. K., and Charkowski, A. O. 2013. The Type III secreted effector DspE is required early in Solanum tuberosum leaf infection by Pectobacterium carotovorum to cause cell death, and requires Wx(3-6)D/E motifs. PLoS One 8(6):e65534 doi:10.1371/journal.pone.0065534.

11. Jacobs, J. M., Babujee, L., Meng, F., Milling, A., and Allen, C. 2012. The in planta transcriptome of Ralstonia solanacearum: Conserved physiological and virulence strategies during bacterial wilt of tomato. MBio. 3(4):e00114-12 doi:mBio.00114-12 [pii] 10.1128/mBio.00114-12.

12. Jeong, Y., Cheong, H., Choi, O., Kim, J. K., Kang, Y., Kim, J., Lee, S., Koh, S., Moon, J. S., and Hwang, I. 2011. An HrpB-dependent but type III-independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum. Mol. Plant Pathol. 12:373-380 doi:10.1111/j.1364-3703.2010.00679.x.

13. Kim, H. S., Thammarat, P., Lommel, S. A., Hogan, C. S., and Charkowski, A. O. 2011. Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions. Mol. Plant Microbe Interact. 24:773-786 doi:10.1094/MPMI-06-10-0143.

14. Kvitko, B. H., Park, D. H., Velásquez, A. C., Wei, C. F., Russell, A. B., Martin, G. B., Schneider, D. J., and Collmer, A. 2009. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 Type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog. 5(4):e1000388 doi:10.1371/journal.ppat.1000388.

15. Milling, A. S., Babujee, L., and Allen, C. 2011. Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PLoS One 6:e15853.

16. Monteiro, F., Genin, S., van Dijk, I., and Valls, M. 2012. A luminescent reporter evidences active expression of Ralstonia solanacearum Type III secretion system genes throughout plant infection. Microbiology 158:2107-2116 doi:mic.0.058610-0 [pii] 10.1099/mic.0.058610-0.

17. Poueymiro, M., and Genin, S. 2009. Secreted proteins from Ralstonia solanacearum: A hundred tricks to kill a plant. Curr. Opin. Microbiol. 12:44-52 doi:S1369-5274(08)00181-1 [pii] 10.1016/j.mib.2008.11.008.

18. Poueymiro, M., Cunnac, S., Barberis, P., Deslandes, L., Peeters, N., Cazale-Noel, A.-C., Boucher, C., and Genin, S. 2009. Two Type III secretion system effectors from Ralstonia solanacearum GMI1000 determine host-range specificity on tobacco. Mol. Plant Microbe Interact. 22:538-550.

19. Prior, P., and Fegan, M. 2005. Recent developments in the phylogeny and classification of Ralstonia solanacearum. Acta Hortic. 695:127-136.

20. Remenant, B., de Cambiaire, J. C., Cellier, G., Jacobs, J. M., Mangenot, S., Barbe, V., Lajus, A., Vallenet, D., Medigue, C., Fegan, M., Allen, C., and Prior, P. 2011. Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles. PLoS One 6(9):e24356 doi:PONE-D-11-07017 [pii] 10.1371/journal.pone.0024356.

21. Remenant, B., Coupat-Goutaland, B., Guidot, A., Cellier, G., Wicker, E., Allen, C., Fegan, M., Pruvost, O., Elbaz, M., Calteau, A., Salvignol, G., Mornico, D., Mangenot, S., Barbe, V., Médigue, C., and Prior, P. 2010. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence. BMC Genomics 11:379.

22. Robertson, A. E., Wechter, W. P., Denny, T., Fortnum, B. A., and Kluepfel, D. A. 2004. Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence. Mol. Plant-Microbe Interact. 17:1376-1384 doi:10.1094/MPMI.2004.17.12.1376.

23. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731-2739 doi:msr121 [pii] 10.1093/molbev/msr121.

24. Thoquet, P., Olivier, J., Sperisen, C., Rogowsky, P., Laterrot, H. and Grimsley, N. 1996. Quantitative trait loci determining resistance to bacterial wilt in tomato cultivar Hawaii7996. Mol. Plant-Microbe Interact. 9:826-836.

25. Yoshimochi, Y., Hikichi, Y., Kiba, A., and Ohnishi, K. 2009. The global virulence regulator PhcA negatively controls the Ralstonia solanacearum hrp regulatory cascade by repressing expression of the PrhIR signaling proteins. J. Bacteriol. 191:3424-3428.