Jonathan M. Jacobs

The In Planta Transcriptome of Ralstonia solanacearum: Conserved Physiological and Virulence Strategies during Bacterial Wilt of Tomato

ABSTRACT Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 108 to 109 CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops. Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.

Ralfuranone Biosynthesis in Ralstonia solanacearum Suggests Functional Divergence in the Quinone Synthetase Family of Enzymes

Ralstonia solanacearum is a destructive crop plant pathogen and produces ralfuranone, i.e., a monophenyl-substituted furanone. Extensive feeding experiments with (13)C-labeled L-phenylalanine now proved that all carbon atoms of the heterocycle derive, after deamination, from this aromatic amino acid. A genetic locus was identified which encodes the aminotransferase RalD and the furanone synthetase RalA. The latter is a tridomain nonribosomal peptide synthetase (NRPS)-like enzyme which was characterized (1) biochemically by the ATP-pyrophosphate exchange assay, and (2) genetically through gene inactivation and transcriptional analysis in axenic culture and in planta. This is the first study to our knowledge on the biochemical and genetic basis of R. solanacearum secondary metabolism. It implies new chemistry for NRPSs, as RalA-mediated biosynthesis requires C-C-bond and subsequent C-O-bond formation to establish the furanone ring system.

The global virulence regulators VsrAD and PhcA control secondary metabolism in the plant pathogen Ralstonia solanacearum.

The soil-borne, Gram-negative bacterium Ralstonia solanacearum is a destructive plant pathogen in both temperate and tropical climates, as it causes a lethal wilt disease. The host range of this organism is remarkably broad and includes plants used for staple food production, such as banana and potato, and species of significant commercial value, including tomato, eggplant, and tobacco. One subgroup of R. solanacearum strains has been classified to contain potential bioterrorism agents, and is subject to rigorous quarantine regulations. Given its relevance as pathogen, R. solanacearum has been thoroughly studied on the genetic, genomic, and physiological level for virulence factors, such as motility and exopolysaccharide (EPS) that have been implicated in bacterial wilt disease progress. 2] However, apart from one report pertaining to biosynthesis and regulation of 3-hydroxy-oxindole, its capacities to synthesize small molecule secondary products have not received much attention yet and are still largely unknown. Here, we report that the global virulence regulatory proteins VsrAD and PhcA affect the biosynthesis of secondary metabolites in R. solanacearum, along with the structure elucidation of ralfuranone (4-phenylfuran2(5H)-one, 1), a novel furanone natural product synthesized by R. solanacearum strain GMI1000. Previous studies revealed that VsrAD and PhcA play central roles in the regulation of diverse functions needed by R. solanacearum during growth inside host plants. We therefore hypothesized that they might also affect expression of secondary metabolites important in survival or virulence in planta. VsrAD is a two-component regulatory system in R. solanacearum that broadly governs expression of many stress and virulence factors. Mutants lacking VsrAD are hypermotile, do not produce EPS, and fail to cause wilt disease on tomato plants. PhcA is a LysR-type global virulence regulator that controls EPS production, bacterial motility, and several other virulence factors in response to a quorum-sensing mechanism. The secondary metabolite profile of wild type R. solanacearum GMI1000 and mutant strains defective in the genes vsrAD (strain RS22) and phcA (strain RS19), respectively, were compared chromatographically. By using HPLC, we identified one major compound in the crude ethyl acetate extract from 4 d old culture supernatants of the wild type strain (1, UV: lmax = 275 nm, m/z 160). This compound eluted after 19.9 min, along with two minor components (m/z 162 and 176, respectively), which eluted after 19.2 and 21.8 min with very similar UV-VIS spectra (lmax = 281 and 277 nm). All these compounds were absent from vsrAD mutant strain RS22 (Figure 1). The ab-

Ralstonia solanacearum Requires PopS, an Ancient AvrE-Family Effector, for Virulence and To Overcome Salicylic Acid-Mediated Defenses during Tomato Pathogenesis

ABSTRACT During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. IMPORTANCE The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens. The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2

While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

Ralfuranone Thioether Production by the Plant Pathogen Ralstonia solanacearum

Ralfuranones are aryl‐substituted furanone secondary metabolites of the Gram‐negative plant pathogen Ralstonia solanacearum. New sulfur‐containing ralfuranone derivatives were identified, including the methyl thioether‐containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L‐methionine or α‐keto‐γ‐methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.

Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco

ABSTRACT Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. IMPORTANCE Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen’s sensitivity to SA toxicity and increases its virulence on tobacco. Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen’s sensitivity to SA toxicity and increases its virulence on tobacco.

Sensitive, Secure Detection of Race 3 Biovar 2 and Native U.S. Strains of Ralstonia solanacearum

Detecting and correctly identifying Ralstonia solanacearum in infected plants is important because the race 3 biovar 2 (R3bv2) subgroup is a high-concern quarantine pathogen, while the related sequevar 7 group is endemic to the southeastern United States. Preventing accidental import of R3bv2 in geranium cuttings demands sensitive detection methods that are suitable for large-volume use both onshore and offshore. However, detection is complicated by frequent asymptomatic latent infections, uneven pathogen distribution within infected plants, pathogen viable-but-not-culturable state, and biosecurity laws that restrict transport of R3bv2 strains for diagnosis. There are many methods to detect R3bv2 strains but their relative utility is unknown, particularly in the realistic context of infected plant hosts. Therefore, we compared the sensitivity, cost, and technical complexity of several assays to detect and distinguish R3bv2 and sequevar 7 strains of R. solanacearum in geranium, tomato, and potato tissue in the laboratory and in naturally infected tomato plants from the field. The sensitivity of polymerase chain reaction (PCR)-based methods in infected geranium tissues was significantly improved by use of Kapa3G Plant, a polymerase with enhanced performance in the presence of plant inhibitors. R3bv2 cells were killed within 60 min of application to Whatman FTA(R) nucleic acid-binding cards, suggesting that samples on FTA cards can be safely transported for diagnosis. Overall, culture enrichment followed by dilution plating was the most sensitive detection method (101 CFU/ml) but it was also most laborious. Conducting PCR from FTA cards was faster, easier, and sensitive enough to detect approximately 104 CFU/ml, levels similar to those found in latently infected geranium plants.