Biology subjects

Carter, M. D.

Publications and source records attributed to Carter, M. D..

4 recordsLinked to original sources

bioRxiv2025

Ralstonia pseudosolanacearum LOV domain protein regulates environmental stress tolerance, iron homeostasis, and bacterial wilt virulence

Ralstonia pseudosolanacearum (Rps), which causes bacterial wilt disease of many crops, must integrate environmental signals to successfully transition from soil to its pathogenic niche in host plant xylem tissue. Mutating a putative sensing/signaling gene had little transcriptomic effect on Rps strain GMI1000 in culture. However, when the mutant grew in tomato over 180 genes were differentially expressed relative to wild type. The gene was therefore named rprR for Ralstonia plant-responsive regulator. In planta, the {Delta}rprR mutant dysregulated genes for diverse traits including stress response, degradation of phenolic compounds, motility, attachment, and production of extracellular polysaccharide (EPS), which is a key bacterial wilt virulence factor. Quantifying Rps EPS by ELISA found increased levels in stems of plants infected with {Delta}rprR as compared to wild type. Functional assays showed {Delta}rprR is defective in attachment to tomato roots, colonization of tomato stems, and bacterial wilt virulence. In rich medium, {Delta}rprR formed biofilm normally, but the mutant formed less biofilm in tomato stem homogenate and in tomato xylem sap under flow. This phenotype correlates with the mutants altered expression of EPS biosynthetic genes and aberrant extracellular matrix. When grown in tomato stem homogenate, {Delta}rprR produced 57% more of the bacterial signal cyclic di-GMP (c-di-GMP) than wild type. This is consistent with the presence in RprR of predicted c-di-GMP modulating domains. Together these findings reveal that RprR, which is highly conserved across plant pathogenic Ralstonia, modulates several bacterial wilt virulence traits in response to the plant host. ImportanceMembers of the Ralstonia solanacearum species complex (RSSC) cause bacterial wilt, a globally destructive disease of market and subsistence crops. Like other plant-associated microbes, bacteria in the RSSC must integrate a complex array of biotic and abiotic signals to successfully infect plant hosts. RSSC genomes all encode an unusual protein, termed RprR, that contains multiple sensing and signaling domains, including two putative modulators of the secondary messenger c-di-GMP. Deleting RprR in Ralstonia pseudosolanacearum had a plant-dependent effect on many traits, including production of the key virulence factors biofilm and exopolysaccharide, as well as intracellular c-di-GMP levels. While c-di-GMP has been investigated in other plant pathogenic bacteria, this is the first report of its role in the RSSC. Most importantly, rprR was required for Ralstonia to effectively colonize plants and cause wilt disease. Thus, RprR is a plant-responsive sensor-regulator that controls pathogen adaptation to the host environment and virulence.

microbiology↗

bioRxiv2025

The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta, we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers "biofilm mobility", which allows wildtype RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia. In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics. Significance StatementRalstonia solanacearum species complex (RSSC) pathogens threaten global food security by fatally wilting plants. A soft matter physics lens demystified the cryptic role of a major virulence factor, the EPS-I exopolysaccharide. EPS-I transforms RSSC biofilms into viscoelastic fluids, a mechanical behavior not previously described for other microbial biofilms that are almost always viscoelastic solids. We demonstrate that the development of fluid biofilms was a key evolutionary innovation that enabled pathogenic success of these aggressive pathogens that rapidly wilt plants.

microbiology↗

bioRxiv2024

Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate- binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a {Delta}lecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the {Delta}lecF/X ECM could explain the double mutants hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment. Author SummaryBacterial wilt diseases caused by Ralstonia species inflict significant losses on diverse, globally important agricultural plants. The pathogen first colonizes roots and ultimately the water-transporting xylem. There it attaches to host cell walls and other bacterial cells to form biofilms that eventually block xylem vessels and disrupt sap flow. It is not well known how Ralstonia spp. modulate attachment, but precise control of both attachment and dispersal is critical for successful host colonization over the disease cycle. Excessive adhesion could trap bacteria in a toxic or nutrient-depleted environment. Conversely, insufficient adhesion in a flowing environment could displace bacteria from an optimal niche. We provide evidence of dual, environment-specific roles of carbohydrate-binding lectins and exopolysaccharide EPS I in Ralstonia pseudosolanacearum (Rps) attachment. In static conditions, which Rps experiences on a host root, two lectins suppress bacterial aggregation and adhesion to roots. However, in flowing conditions, which Rps experiences in healthy xylem vessels, the same two lectins and EPS I are essential for biofilm development. The lectins increase the biofilm viscosity and support colony structural integrity, likely by interacting with polysaccharides in the biofilm matrix. This novel multifunctionality of bacterial lectins reveals how pathogens adapt to a physically dynamic host environment.

microbiology↗

bioRxiv2022

Cell-density regulated adhesins contribute to early disease development and adhesion in Ralstonia solanacearum

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum (Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a {Delta}phcA quorum sensing mutant that cannot respond to high cell densities: radA (Ralstonia adhesin), rcpA (Ralstonia collagen-like protein), and rcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization Rs upregulated rcpA and rcpB, but not radA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with {Delta}rcpA/B and {Delta}radA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. ImportanceEvery microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of several afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three individual plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to bacterial success. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis. Research AreasPlant Microbiology, Host-Microbial Interactions, Microbial Pathogenesis

microbiology↗