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Scholarly Works (8 results)

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UC Davis Electronic Theses and Dissertations (2021)

Bacterial pathogens in the Ralstonia solanacearum species complex cause widespread bacterial wilt diseases throughout the humid tropics and subtropics, causing significant damage to numerous susceptible crop species. Ralstonia are diverse plant pathogenic bacteria with thousands of distinct lineages. DNA sequence analysis reveals the species complex is comprised of four primary branches (phylotypes) of continental or archipelagic origins in Africa, Asia, the Americas, and the tropical islands of the South Pacific. The Ralstonia pathogen family has a remarkably broad host range, infecting hundreds of host species in dozens of taxonomic families (Álvarez 2010). This breadth, however, is not uniformly distributed across strains. The IIB-4 clade is known to exhibit a particularly broad host range. When introduced to new geographic locations, IIB-4 strains can proliferate rapidly and infect crop species not susceptible to previously endemic strains (Wicker 2007).

In this investigation, we quantified the virulence and host range of 19 strains in the IIB-4 clade through stem and soil inoculations on four diverse host species: tomato (Solanum lycopersicum cv Moneymaker), melon (Cucumis melo cv. Sweet Granite), Impatiens (Impatiens walleriana cv. Beacon Orange), and banana (Musa acuminata cv. Dwarf Cavendish). The strains we analyzed originate from a broad geographic swath of the ancestral range of phylotype II, including Florida, the Caribbean, Central America, and South America. 18 strains were isolated from monocotyledonous plantain (Musa spp.), Heliconia (Heliconia spp.), pothos (Epipremnum aureum), and Anthurium (Anthurium spp.) plants. One strain (IBSBF1503) is a eudicot isolate from cucumber (Cucumis sativus). To differentiate patterns of host specificity and virulence amongst the closely related strains, we subjected each host-strain combination to disease progress assays in a controlled growth chamber environment. We calculated area under disease progress curve (AUDPC) values individually for each of the 3,005 plants in the study and aggregately for each host-strain combination. Highly virulent strains caused disease progressions with mean AUDPC values of 8  MAUDPC. Moderately virulent strains generated mean AUDPC values of 4  MAUDPC < 8. Strains with low virulence generated mean AUDPC values of 0  MAUDPC < 4. Avirulent strains generated mean AUDPC values of MAUDPC = 0.

Tomato plants were the most broadly susceptible. 17 strains were highly virulent on tomato plants, and two strains were moderately virulent. Virulence on melon plants was more variable. Eleven strains were highly virulent on melon plants, four strains were moderately virulent, and four strains exhibited low virulence. Virulence on Impatiens plants was also variable. Twelve strains were highly virulent on Impatiens plants, four strains were moderately virulent, and three strains exhibited low virulence. Banana plants were the most broadly resistant. One strain was highly virulent on banana plants, two strains were moderately virulent, five strains exhibited low virulence, and eleven strains were avirulent. Despite a high degree of genetic similarity, R. solanacearum strains in the IIB/4 clade exhibit considerable variation in virulence and pathogenicity on diverse hosts.

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UC Davis Previously Published Works (2017)
The PhcA virulence regulator in the vascular wilt pathogen Ralstonia solanacearum responds to cell density via quorum sensing. To understand the timing of traits that enable R. solanacearum to establish itself inside host plants, we created a ΔphcA mutant that is genetically locked in a low-cell-density condition. Comparing levels of gene expression of wild-type R. solanacearum and the ΔphcA mutant during tomato colonization revealed that the PhcA transcriptome includes an impressive 620 genes (>2-fold differentially expressed; false-discovery rate [FDR], ≤0.005). Many core metabolic pathways and nutrient transporters were upregulated in the ΔphcA mutant, which grew faster than the wild-type strain in tomato xylem sap and on dozens of specific metabolites, including 36 found in xylem. This suggests that PhcA helps R. solanacearum to survive in nutrient-poor environmental habitats and to grow rapidly during early pathogenesis. However, after R. solanacearum reaches high cell densities in planta, PhcA mediates a trade-off from maximizing growth to producing costly virulence factors. R. solanacearum infects through roots, and low-cell-density-mode-mimicking ΔphcA cells attached to tomato roots better than the wild-type cells, consistent with their increased expression of several adhesins. Inside xylem vessels, ΔphcA cells formed aberrantly dense mats. Possibly as a result, the mutant could not spread up or down tomato stems as well as the wild type. This suggests that aggregating improves R. solanacearum survival in soil and facilitates infection and that it reduces pathogenic fitness later in disease. Thus, PhcA mediates a second strategic switch between initial pathogen attachment and subsequent dispersal inside the host. PhcA helps R. solanacearum optimally invest resources and correctly sequence multiple steps in the bacterial wilt disease cycle.IMPORTANCERalstonia solanacearum is a destructive soilborne crop pathogen that wilts plants by colonizing their water-transporting xylem vessels. It produces its costly virulence factors only after it has grown to a high population density inside a host. To identify traits that this pathogen needs in other life stages, we studied a mutant that mimics the low-cell-density condition. This mutant (the ΔphcA mutant) cannot sense its own population density. It grew faster than and used many nutrients not available to the wild-type bacterium, including metabolites present in tomato xylem sap. The mutant also attached much better to tomato roots, and yet it failed to spread once it was inside plants because it was trapped in dense mats. Thus, PhcA helps R. solanacearum succeed over the course of its complex life cycle by ensuring avid attachment to plant surfaces and rapid growth early in disease, followed by high virulence and effective dispersal later in disease.
    Cover page: A Single Regulator Mediates Strategic Switching between Attachment/Spread and Growth/Virulence in the Plant Pathogen Ralstonia solanacearum
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    UC Davis Previously Published Works (2018)
    Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant-like expansin genes in numerous xylem-colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non-chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem-inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β-proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm ΔCmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the ΔCmEXLX2 mutant, even though the strains grew as well as the wild-type in vitro. Similarly, when inoculated onto tomato fruit, ΔCmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs ΔRsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, ΔRsEXLX attached to tomato seedling roots better than the wild-type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non-chimeric bacterial expansins and highlight their importance in plant-bacterial interactions.
      Cover page: Plant‐like bacterial expansins play contrasting roles in two tomato vascular pathogens
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      UC Davis Previously Published Works (2016)
      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. 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.
        Cover page: Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco
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        UC Davis Previously Published Works (2022)
        Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem tissue and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures and physiological traits. To increase our understanding of the evolution, diversity and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4940 genes on average, and a pangenome analysis found that there are 3262 genes in the core genome (~60 % of the mean RSSC genome) with 13 128 genes in the extensive flexible genome. A core genome phylogenetic tree and a whole-genome similarity matrix aligned with the previously named species ( R. solanacearum , R. pseudosolanacearum , R. syzygii ) and phylotypes (I–IV). These analyses also highlighted a third unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. We also used our genome-based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved, cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome-based identification of newly sequenced RSSC strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.
          Cover page: Meta-analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology
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          UC Davis Previously Published Works (2021)
          Plant-pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens [RB-TnSeq]) in three strains spanning the genetic, geographical, and physiological range of plant-pathogenic Ralstonia: Ralstonia solanacearum IBSBF1503, Ralstonia pseudosolanacearum GMI1000, and Ralstonia syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and lipopolysaccharide (LPS) biosynthesis. Mutant strains with mutations in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains of fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition. IMPORTANCE Traditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here, we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of the wild type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.
            Cover page: Genome-Wide Identification of Tomato Xylem Sap Fitness Factors for Three Plant-Pathogenic Ralstonia Species
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            UC Davis Previously Published Works (2018)
            Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite.
              Cover page: Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease
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              UC Davis Previously Published Works (2023)
              Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.
                Cover page: Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem
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