Transcriptome sequencing and expression analysis in peanut reveal the potential mechanism response to Ralstonia solanacearum infection.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum severely affects peanut (Arachis hypogaea L.) yields. The breeding of resistant cultivars is an efficient means of controlling plant diseases. Therefore, identification of resistance genes effective against bacterial wilt is a matter of urgency. The lack of a reference genome for a resistant genotype severely hinders the process of identification of resistance genes in peanut. In addition, limited information is available on disease resistance-related pathways in peanut. RESULTS: Full-length transcriptome data were used to generate wilt-resistant and -susceptible transcript pools. In total, 253,869 transcripts were retained to form a reference transcriptome for RNA-sequencing data analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes revealed the plant-pathogen interaction pathway to be the main resistance-related pathway for peanut to prevent bacterial invasion and calcium plays an important role in this pathway. Glutathione metabolism was enriched in wilt-susceptible genotypes, which would promote glutathione synthesis in the early stages of pathogen invasion. Based on our previous quantitative trait locus (QTL) mapping results, the genes arahy.V6I7WA and arahy.MXY2PU, which encode nucleotide-binding site-leucine-rich repeat receptor proteins, were indicated to be associated with resistance to bacterial wilt. CONCLUSIONS: This study identified several pathways associated with resistance to bacterial wilt and identified candidate genes for bacterial wilt resistance in a major QTL region. These findings lay a foundation for investigation of the mechanism of resistance to bacterial wilt in peanut.

Plant pathogenic bacterium Ralstonia solanacearum can rapidly evolve tolerance to antimicrobials produced by Pseudomonas biocontrol bacteria.

Soil-borne plant pathogens significantly threaten crop production due to lack of effective control methods. One alternative to traditional agrochemicals is microbial biocontrol, where pathogen growth is suppressed by naturally occurring bacteria that produce antimicrobial chemicals. However, it is still unclear if pathogenic bacteria can evolve tolerance to biocontrol antimicrobials and if this could constrain the long-term efficacy of biocontrol strategies. Here we used an in vitro experimental evolution approach to investigate if the phytopathogenic Ralstonia solanacearum bacterium, which causes bacterial wilt disease, can evolve tolerance to antimicrobials produced by Pseudomonas bacteria. We further asked if tolerance was specific to pairs of R. solanacearum and Pseudomonas strains and certain antimicrobial compounds produced by Pseudomonas. We found that while all R. solanacearum strains could initially be inhibited by Pseudomonas strains, this inhibition decreased following successive subculturing with or without Pseudomonas supernatants. Using separate tolerance assays, we show that the majority of R. solanacearum strains evolved increased tolerance to multiple Pseudomonas strains. Mechanistically, evolved tolerance was most likely linked to reduced susceptibility to orfamide lipopeptide antimicrobials secreted by Pseudomonas strains in our experimental conditions. Some levels of tolerance also evolved in the control treatments, which was likely correlated response due to adaptations to the culture media. Together, these results suggest that plant-pathogenic bacteria can rapidly evolve increased tolerance to bacterial antimicrobial compounds, which could reduce the long-term efficacy of microbial biocontrol.

Rastonia solanacearum type â ¢ effectors target host 14-3-3 proteins to suppress plant immunity.

14-3-3 proteins play important roles in plant metabolism and stress response. Tomato 14-3-3 proteins, SlTFT4 and SlTFT7, serve as hubs of plant immunity and are targeted by some pathogen effectors. Ralstonia solanacearum with more than 70 type Ⅲ effectors (T3Es) is one of the most destructive plant pathogens. However, little is known on whether R. solanacearum T3Es target SlTFT4 and SlTFT7 and hence interfere with plant immunity. We first detected the associations of SlTFT4/SlTFT7 with R. solanacearum T3Es by luciferase complementation assay, and then confirmed the interactions by yeast two-hybrid approach. We demonstrated that 22 Ralstonia T3Es were associated with both SlTFT4 and SlTFT7, and five among them suppressed the hypersensitive response induced by MAPKKKα, a protein kinase which associated with SlTFT4/SlTFT7. We further demonstrated that suppression of MAPKKKα-induced HR and plant basal defense by the T3E RipAC depend on its association with 14-3-3 proteins. Our findings firstly demonstrate that R. solanacearum T3Es can manipulate plant immunity by targeting 14-3-3 proteins, SlTFT4 and SlTFT7, providing new insights into plant-R. solanacearum interactions.

Ralstonia solanacearum effector RipAK suppresses homodimerization of the host transcription factor ERF098 to enhance susceptibility and the sensitivity of pepper plants to dehydration.

Plants have evolved a sophisticated immune system to defend against invasion by pathogens. In response, pathogens deploy copious effectors to evade the immune responses. However, the molecular mechanisms used by pathogen effectors to suppress plant immunity remain unclear. Herein, we report that an effector secreted by Ralstonia solanacearum, RipAK, modulates the transcriptional activity of the ethylene-responsive factor ERF098 to suppress immunity and dehydration tolerance, which causes bacterial wilt in pepper (Capsicum annuum L.) plants. Silencing ERF098 enhances the resistance of pepper plants to R. solanacearum infection not only by inhibiting the host colonization of R. solanacearum but also by increasing the immunity and tolerance of pepper plants to dehydration and including the closure of stomata to reduce the loss of water in an abscisic acid signal-dependent manner. In contrast, the ectopic expression of ERF098 in Nicotiana benthamiana enhances wilt disease. We also show that RipAK targets and inhibits the ERF098 homodimerization to repress the expression of salicylic acid-dependent PR1 and dehydration tolerance-related OSR1 and OSM1 by cis-elements in their promoters. Taken together, our study reveals a regulatory mechanism used by the R. solanacearum effector RipAK to increase virulence by specifically inhibiting the homodimerization of ERF098 and reprogramming the transcription of PR1, OSR1, and OSM1 to boost susceptibility and dehydration sensitivity. Thus, our study sheds light on a previously unidentified strategy by which a pathogen simultaneously suppresses plant immunity and tolerance to dehydration by secreting an effector to interfere with the activity of a transcription factor and manipulate plant transcriptional programs.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Uncovering the transcriptional responses of tobacco (Nicotiana tabacum L.) roots to Ralstonia solanacearum infection: a comparative study of resistant and susceptible cultivars.

BACKGROUND: Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious soil-borne disease of tobacco that significantly reduces crop yield. However, the limited availability of resistance in tobacco hinders breeding efforts for this disease. RESULTS: In this study, we conducted hydroponic experiments for the root expression profiles of D101 (resistant) and Honghuadajinyuan (susceptible) cultivars in response to BW infection at 0 h, 6 h, 1 d, 3 d, and 7d to explore the defense mechanisms of BW resistance in tobacco. As a result, 20,711 and 16,663 (total: 23,568) differentially expressed genes (DEGs) were identified in the resistant and susceptible cultivars, respectively. In brief, at 6 h, 1 d, 3 d, and 7 d, the resistant cultivar showed upregulation of 1553, 1124, 2583, and 7512 genes, while the susceptible cultivar showed downregulation of 1213, 1295, 813, and 7735 genes. Similarly, across these time points, the resistant cultivar had downregulation of 1034, 749, 1686, and 11,086 genes, whereas the susceptible cultivar had upregulation of 1953, 1790, 2334, and 6380 genes. The resistant cultivar had more up-regulated genes at 3 d and 7 d than the susceptible cultivar, indicating that the resistant cultivar has a more robust defense response against the pathogen. The GO and KEGG enrichment analysis showed that these genes are involved in responses to oxidative stress, plant-pathogen interactions, cell walls, glutathione and phenylalanine metabolism, and plant hormone signal transduction. Among the DEGs, 239 potential candidate genes were detected, including 49 phenylpropane/flavonoids pathway-associated, 45 glutathione metabolic pathway-associated, 47 WRKY, 48 ERFs, eight ARFs, 26 pathogenesis-related genes (PRs), and 14 short-chain dehydrogenase/reductase genes. In addition, two highly expressed novel genes (MSTRG.61386-R1B-17 and MSTRG.61568) encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins were identified in both cultivars at 7 d. CONCLUSIONS: This study revealed significant enrichment of DEGs in GO and KEGG terms linked to glutathione, flavonoids, and phenylpropane pathways, indicating the potential role of glutathione and flavonoids in early BW resistance in tobacco roots. These findings offer fundamental insight for further exploration of the genetic architecture and molecular mechanisms of BW resistance in tobacco and solanaceous plants at the molecular level.

StMLP1, as a Kunitz trypsin inhibitor, enhances potato resistance and specifically expresses in vascular bundles during Ralstonia solanacearum infection.

Miraculin-like proteins (MLPs), members of the Kunitz trypsin inhibitor (KTI) family that are present in various plants, have been discovered to have a role in defending plants against pathogens. In this study, we identified a gene StMLP1 in potato that belongs to the KTI family. We found that the expression of StMLP1 gradually increases during Ralstonia solanacearum (R. solanacearum) infection. We characterized the promoter of StMLP1 as an inducible promoter that can be triggered by R. solanacearum and as a tissue-specific promoter with specificity for vascular bundle expression. Our findings demonstrate that StMLP1 exhibits trypsin inhibitor activity, and that its signal peptide is essential for proper localization and function. Overexpression of StMLP1 in potato can enhance the resistance to R. solanacearum. Inhibiting the expression of StMLP1 during infection accelerated the infection by R. solanacearum to a certain extent. In addition, the RNA-seq results of the overexpression-StMLP1 lines indicated that StMLP1 was involved in potato immunity. All these findings in our study reveal that StMLP1 functions as a positive regulator that is induced and specifically expressed in vascular bundles in response to R. solanacearum infection.

Quorum Sensing-Dependent Invasion of Ralstonia solanacearum into Fusarium oxysporum Chlamydospores.

Strains of the Ralstonia solanacearum species complex (RSSC), although known as the causative agent of bacterial wilt disease in plants, induce the chlamydospores of many fungal species and invade them through the spores. The lipopeptide ralstonins are the chlamydospore inducers produced by RSSC and are essential for this invasion. However, no mechanistic investigation of this interaction has been conducted. In this study, we report that quorum sensing (QS), which is a bacterial cell-cell communication, is important for RSSC to invade the fungus Fusarium oxysporum (Fo). ΔphcB, a deletion mutant of QS signal synthase, lost the ability to both produce ralstonins and invade Fo chlamydospores. The QS signal methyl 3-hydroxymyristate rescued these disabilities. In contrast, exogenous ralstonin A, while inducing Fo chlamydospores, failed to rescue the invasive ability. Gene-deletion and -complementation experiments revealed that the QS-dependent production of extracellular polysaccharide I (EPS I) is essential for this invasion. The RSSC cells adhered to Fo hyphae and formed biofilms there before inducing chlamydospores. This biofilm formation was not observed in the EPS I- or ralstonin-deficient mutant. Microscopic analysis showed that RSSC infection resulted in the death of Fo chlamydospores. Altogether, we report that the RSSC QS system is important for this lethal endoparasitism. Among the factors regulated by the QS system, ralstonins, EPS I, and biofilm are important parasitic factors. IMPORTANCE Ralstonia solanacearum species complex (RSSC) strains infect both plants and fungi. The phc quorum-sensing (QS) system of RSSC is important for parasitism on plants, because it allows them to invade and proliferate within the hosts by causing appropriate activation of the system at each infection step. In this study, we confirm that ralstonin A is important not only for Fusarium oxysporum (Fo) chlamydospore induction but also for RSSC biofilm formation on Fo hyphae. Extracellular polysaccharide I (EPS I) is also essential for biofilm formation, while the phc QS system controls these factors in terms of production. The present results advocate a new QS-dependent mechanism for the process by which a bacterium invades a fungus.

Genome-Wide Identification of the NPR1-like Gene Family in Solanum tuberosum and Functional Characterization of StNPR1 in Resistance to Ralstonia solanacearum.

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon-intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon-intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, ß-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum.

Protein phosphatase StTOPP6 negatively regulates potato bacterial wilt resistance by modulating MAPK signaling.

Potato (Solanum tuberosum) is an important crop globally and is grown across many regions in China, where it ranks fourth in the list of staple foods. However, its production and quality are severely affected by bacterial wilt caused by Ralstonia solanacearum. In this study, we identified StTOPP6, which belongs to the type one protein phosphatase (TOPP) family, and found that transient knock down of StTOPP6 in potato increased resistance against R. solanacearum. RNA-seq analysis showed that knock down of StTOPP6 activated immune responses, and this defense activation partly depended on the mitogen-activated protein kinase (MAPK) signal pathway. StTOPP6 inhibited the expression of StMAPK3, while overexpression of StMAPK3 enhanced resistance to R. solanacearum, supporting the negative role of StTOPP6 in plant immunity. Consistent with the results of knock down of StTOPP6, overexpressing the phosphatase-dead mutation StTOPP6m also attenuated infection and up-regulated MAPK3, showing that StTOPP6 activity is required for disease. Furthermore, we found that StTOPP6 affected the StMAPK3-mediated downstream defense pathway, eventually suppressing the accumulation of reactive oxygen species (ROS). Consistent with these findings, plants with knock down of StTOPP6, overexpression of StTOPP6m, and overexpression of StMAPK3 all displayed ROS accumulation and enhanced resistance to R. solanacearum. Taken together, the findings of our study demonstrate that StTOPP6 negatively regulates resistance to bacterial wilt by affecting the MAPK3-mediated pathway.

MADS-box protein AGL8 interacts with chromatin-remodelling component SWC4 to activate thermotolerance and environment-dependent immunity in pepper.

Pepper (Capsicum annuum) employs distinct defence responses against Ralstonia solanacearum infection (RSI); however, the mechanisms by which pepper activates these defence responses in a context-dependent manner is unclear. Here we study pepper plants defence response to RSI under room temperature-high humidity (RSRT, 28 °C / 90%) and high temperature-high humidity (RSHT, 37 °C / 90%) conditions, and non-infected plants under high temperature-high humidity (HTHH, 42 °C / 90%) stress. Herein, we found that the MADS-box transcription factor CaAGL8 was up-regulated by HTHH stress and RSRT or RSHT, and its silencing significantly reduced pepper thermotolerance and susceptibility to infection under both room and high temperature-high humidity (RSRT and RSHT). This was coupled with down-regulation of CaSTH2 and CaDEF1 upon RSRT, down-regulation of CaMgst3 and CaPRP1 upon RSHT, and down-regulation of CaHSP24 upon HTHH. In contrast, the ectopic overexpression of CaAGL8 significantly increased the resistance of Nicotiana benthamiana plants to RSRT, RSHT, and HTHH. In addition, CaAGL8 was found to interact with CaSWC4, which acted as a positive regulator of the pepper response to RSRT, RSHT, and HTHH. Silencing of either CaAGL8 or CaSWC4 blocked the hypersensitive response (HR) cell death and context-dependent up-regulation of defence-related genes triggered by the other. Importantly, enrichment of H4K5Ac, H3K9Ac, H3K4me3, and H3K9me2 on the tested defence-related genes was context- and gene-specifically regulated through synergistic interaction between CaSWC4 and CaAGL8. Our results indicate that pepper employs CaAGL8 to modulate chromatin remodelling by interacting with CaSWC4, thereby activating defence responses to RSRT, RSHT, and HTHH.

Antagonistic Activity of Volatile Organic Compounds Produced by Acid-Tolerant Pseudomonas protegens CLP-6 as Biological Fumigants To Control Tobacco Bacterial Wilt Caused by Ralstonia solanacearum.

Tobacco bacterial wilt, which is caused by Ralstonia solanacearum, is a devastating soilborne disease of tobacco worldwide and is widespread in the continuously acidic fields of southern China. Here, the fumigation activity under different pH conditions, component identification, and bioactivity of the volatile organic compounds (VOCs) produced by an acid-tolerant strain, Pseudomonas protegens CLP-6, were investigated. There was a wide antimicrobial spectrum of the VOCs against phytopathogens, including four bacteria, eight fungi, and two oomycetes. The antagonistic activity of the VOCs against R. solanacearum was proportionally correlated with the concentration of the inoculum, amount, culture time, and culture pH for CLP-6. The number of gene copies of R. solanacearum was significantly inhibited by VOCs produced at pH 5.5 in vivo. The control effect of VOCs emitted at pH 5.5 was 78.91% for tobacco bacterial wilt, which was >3-fold greater than that at pH 7.0. Finally, the main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography-mass spectroscopy (GC-MS) as S-methyl thioacetate, methyl thiocyanate, methyl disulfide, 1-decene, 2-ethylhexanol, 1,4-undecadiene, 1-undecene, 1,3-benzothiazole, and 2,5-dimethylpyrazine, and the inhibition rates of 1,3-benzothiazole, 2-ethylhexanolmethyl thiocyanate, dimethyl disulfide, and S-methyl thioacetate were 100%, 100%, 88.91%, 67.64%, and 53.29%, respectively. S-Methyl thioacetate was detected only at pH 5.5. In summary, VOCs produced by P. protegens CLP-6 had strong antagonistic activities against phytopathogens, especially R. solanacearum, under acidic conditions and could be used to develop a safe and additive fumigant against R. solanacearum on tobacco and even other Solanaceae crop bacterial wilt diseases in acidic fields. IMPORTANCE VOCs produced by beneficial bacteria penetrate the rhizosphere to inhibit the growth of plant-pathogenic microorganisms; thus, they have the potential to be used as biological agents in controlling plant diseases. Tobacco bacterial wilt, which is caused by the acidophilic pathogen R. solanacearum, is a major bacterial disease in southern China and is prevalent in acidic soil. In this study, we discovered that the VOCs produced by P. protegens CLP-6 had excellent inhibitory effects on important plant pathogens. Moreover, two of the VOCs, namely, 1,3-benzothiazole and 2-ethylhexanol, had excellent inhibitory effect on R. solanacearum, and another VOC substance, methyl thiocyanate, was produced only at pH 5.5. The VOCs produced by the acid-tolerant strain P. protegens CLP-6 may have potential as environment-friendly microbial fumigant agents for bacterial wilt of tobacco or even other Solanaceae crops in acidic soils in China.

The Endophytic Root Microbiome Is Different in Healthy and Ralstonia solanacearum-Infected Plants and Is Regulated by a Consortium Containing Beneficial Endophytic Bacteria.

Plant bacterial wilt disease caused by Ralstonia solanacearum leads to huge economic losses worldwide. Endophytes play vital roles in promoting plant growth and health. It is hypothesized that the endophytic root microbiome and network structure are different in healthy and diseased plants. Here, the endophytic root microbiomes and network structures of healthy and diseased tobacco plants were investigated. Composition and network structures of endophytic root microbiomes were distinct between healthy and diseased plants. Healthy plants were enriched with more beneficial bacteria and bacteria with antagonistic activity against R. solanacearum. R. solanacearum was most abundant in diseased plants. Microbial networks in diseased plants had fewer modules and edges, lower connectivity, and fewer keystone microorganisms than those in healthy plants. Almost half of the nodes were unique in the two networks. Ralstonia was identified as a key microorganism of the diseased-plant network. In healthy plants, abundant bacteria and biomarkers (Pseudomonas and Streptomyces) and keystone microorganisms (Bacillus, Lysobacter, and Paenibacillus) were plant-beneficial bacteria and showed antibacterial and plant growth-promoting activities. The endophytic strain Bacillus velezensis E9 produced bacillaene to inhibit R. solanacearum. Consortia containing keystone microorganisms and beneficial endophytic bacteria significantly regulated the endophytic microbiome and attenuated bacterial wilt by inducing systemic resistance and producing antibiotic. Overall, the endophytic root microbiome and network structure in diseased plants were different from those in healthy plants. The endophytic root microbiome of diseased plants had low abundances of beneficial bacteria and an unstable network and lacked beneficial keystone microorganisms, which favored infection. Synthetic microbial consortia were effective measures for preventing R. solanacearum infection. IMPORTANCE Bacterial wilt disease causes heavy yield losses in many crops. Endophytic microbiomes play important roles in control of plant diseases. However, the role of the endophytic root microbiome in controlling bacterial wilt disease is poorly understood. Here, differences in endophytic root microbiomes and network structures between healthy and diseased tobacco plants are reported. A synthetic microbial consortium containing beneficial endophytic bacteria was used to regulate the endophytic microbiome and attenuate bacterial wilt disease. The results could be generally used to guide control of bacterial wilt disease.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

The CaPti1-CaERF3 module positively regulates resistance of Capsicum annuum to bacterial wilt disease by coupling enhanced immunity and dehydration tolerance.

Bacterial wilt, a severe disease involving vascular system blockade, is caused by Ralstonia solanacearum. Although both plant immunity and dehydration tolerance might contribute to disease resistance, whether and how they are related remains unclear. Herein, we showed that immunity against R. solanacearum and dehydration tolerance are coupled and regulated by the CaPti1-CaERF3 module. CaPti1 and CaERF3 are members of the serine/threonine protein kinase and ethylene-responsive factor families, respectively. Expression profiling revealed that CaPti1 and CaERF3 were upregulated by R. solanacearum inoculation, dehydration stress, and exogenously applied abscisic acid (ABA). They in turn phenocopied each other in promoting resistance of pepper (Capsicum annuum) to bacterial wilt not only by activating salicylic acid-dependent CaPR1, but also by activating dehydration tolerance-related CaOSM1 and CaOSR1 and inducing stomatal closure to reduce water loss in an ABA signaling-dependent manner. Our yeast two hybrid assay showed that CaERF3 interacted with CaPti1, which was confirmed using co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that upon R. solanacearum inoculation, CaPR1, CaOSM1, and CaOSR1 were directly targeted and positively regulated by CaERF3 and potentiated by CaPti1. Additionally, our data indicated that the CaPti1-CaERF3 complex might act downstream of ABA signaling, as exogenously applied ABA did not alter regulation of stomatal aperture by the CaPti1-CaERF3 module. Importantly, the CaPti1-CaERF3 module positively affected pepper growth and the response to dehydration stress. Collectively, the results suggested that immunity and dehydration tolerance are coupled and positively regulated by CaPti1-CaERF3 in pepper plants to enhance resistance against R. solanacearum.

Host-specific activation of a pathogen effector Aave_4606 from Acidovorax citrulli, the causal agent for bacterial fruit blotch.

RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.

Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia solanacearum Bacteriophages after Their Conservation Prior to Commercialization and Use.

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titers throughout the experimental period, and with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

Trophic preferences of the pathogen Ralstonia solanacearum and consequences on its growth in xylem sap.

Ralstonia solanacearum is one of the most destructive pathogens worldwide. In the last 30 years, the molecular mechanisms at the origin of R. solanacearum pathogenicity have been studied in depth. However, the nutrition status of the pathogen once inside the plant has been poorly investigated. Yet, the pathogen needs substrates to sustain a fast-enough growth, maintain its virulence and subvert the host immunity. This study aimed to explore in-depth the xylem environment where the pathogen is abundant, and its trophic preferences. First, we determined the composition of tomato xylem sap, where fast multiplication of the pathogen occurs. Then, kinetic growth on single and mixtures of carbon sources in relation to this environment was performed to fully quantify growth. Finally, we calculated the concentration of available metabolites in the xylem sap flux to assess how much it can support bacterial growth in planta. Overall, the study underlines the adaptation of R. solanacearum to the xylem environment and the fact that the pathogen assimilates several substrates at the same time in media composed of several carbon sources. It also provides metrics on key physiological parameters governing the growth of this major pathogen, which will be instrumental in the future to better understand its metabolic behavior during infection.

Solanaceous plants switch to cytokinin-mediated immunity against Ralstonia solanacearum under high temperature and high humidity.

Plant diseases generally tend to be more serious under conditions of high temperature and high humidity (HTHH) than under ambient temperature, but plant immunity against pathogen attacks under HTHH remains elusive. Herein, we used pepper as an example to study how Solanaceae cope with Ralstonia solanacearum infection (RSI) under HTHH by performing RNA-seq combined with the reverse genetic method. The result showed that immunities mediated by salicylic acid (SA) and jasmonic acid (JA) in pepper roots were activated by RSI under ambient temperature. However, upon RSI under HTHH, JA signalling was blocked and SA signalling was activated early but its duration was greatly shortened in pepper roots, instead, expression of CaIPT5 and Glutathione S-transferase encoding genes, as well as endogenous content of trans-Zeatin, were enhanced. In addition, by silencing in pepper plants and overexpression in Nicotiana benthamiana, CaIPT5 was found to act positively in the immune response to RSI under HTHH in a way related to CaPRP1 and CaMgst3. Furthermore, the susceptibility of pepper, tomato and tobacco to RSI under HTHH was significantly reduced by exogenously applied tZ, but not by either SA or MeJA. All these data collectively suggest that pepper employs cytokinin-mediated immunity to cope with RSI under HTHH.

WIPK-NtLTP4 pathway confers resistance to Ralstonia solanacearum in tobacco.

KEY MESSAGE: WIPK-NtLTP4 module improves the resistance to R. solanacearum via upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure in tobacco. Lipid transfer proteins (LTPs) are a class of small lipid binding proteins that play important roles in biotic and abiotic stresses. The previous study revealed that NtLTP4 positively regulates salt and drought stresses in Nicotiana tabacum. However, the role of NtLTP4 in biotic stress, especially regarding its function in disease resistance remains unclear. Here, the critical role of NtLTP4 in regulating resistance to Ralstonia solanacearum (R. solanacearum), a causal agent of bacterial wilt disease in tobacco, was reported. The NtLTP4-overexpressing lines markedly improved the resistance to R. solanacearum by upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure. Moreover, NtLTP4 interacted with wound-induced protein kinase (WIPK; a homolog of MAPK3 in tobacco) and acted in a genetically epistatic manner to WIPK in planta. WIPK could directly phosphorylate NtLTP4 to positively regulate its protein abundance. Taken together, these results broaden the knowledge about the functions of the WIPK-NtLTP4 module in disease resistance and may provide valuable information for improving tobacco plant tolerance to R. solanacearum.