Navigating the signaling landscape of Ralstonia solanacearum: a study of bacterial two-component systems.

Ralstonia solanacearum, the bacterium that causes bacterial wilt, is a destructive phytopathogen that can infect over 450 different plant species. Several agriculturally significant crop plants, including eggplant, tomato, pepper, potato, and ginger, are highly susceptible to this plant disease, which has a global impact on crop quality and yield. There is currently no known preventive method that works well for bacterial wilt. Bacteria use two-component systems (TCSs) to sense their environment constantly and react appropriately. This is achieved by an extracellular sensor kinase (SK) capable of sensing a suitable signal and a cytoplasmic response regulator (RR) which gives a downstream response. Moreover, our investigation revealed that R. solanacearum GMI1000 possesses a substantial count of TCSs, specifically comprising 36 RRs and 27 SKs. While TCSs are known targets for various human pathogenic bacteria, such as Salmonella, the role of TCSs in R. solanacearum remains largely unexplored in this context. Notably, numerous inhibitors targeting TCSs have been identified, including GHL (Gyrase, Hsp, and MutL) compounds, Walk inhibitors, and anti-TCS medications like Radicicol. Consequently, the investigation into the involvement of TCSs in virulence and pathogenesis has gained traction; however, further research is imperative to ascertain whether TCSs could potentially supplant conventional anti-wilt therapies. This review delves into the prospective utilization of TCSs as an alternative anti-wilt therapy, focusing on the lethal phytopathogen R. solanacearum.

Comparative identification of WRKY transcription factors and transcriptional response to Ralstonia solanacearum in tomato.

In order to study the responses of tomato (Solanum lycopersicum) WRKY TFs to bacterial wilt caused by Ralstonia solanacearum, the most up-to-date genomes and transcriptional profiles were used to identify WRKY TFs in control and infected inbred lines. In total, 85 tomato WRKY TFs were identified and categorized into groups I, IIa + b, IIc, IId + e, and III. These WRKYs, especially those from group IIe, were mainly distributed at chromosome ends and in clusters. More than 45 % and 70 % of tomato WRKYs exhibited intraspecific and interspecific synteny, respectively. Nearly 60 % of tomato WRKYs (mainly in groups I and IIc) formed 73 pairs of orthologs with WRKYs in Arabidopsis and pepper, with Ka/Ks less than 1. Sixteen tomato WRKYs (mainly in groups IIa + b and IIc) responded strongly to biotic stress, and 12 differentially expressed WRKYs (mainly in groups III and IIb) were identified. RT-qPCR revealed that tomato WRKYs could respond to bacterial wilt through positive (predominant) or negative regulation. In particular, the interaction between Solyc03g095770.3 (group III) and Solyc09g014990.4 (group I) may play an important role. In brief, WRKY TFs were comprehensively identified in tomato and several bacterial wilt responsive genes were screened.

Pepper defense against Ralstonia solanacearum and High-temperature stress is positively regulated by CaMYB59.

We have functionally evaluated a transcription factor CaMYB59 for its role in pepper immune responses to Ralstonia solanacearum attack and high temperature-high humidity (HTHH). Exposure to R. solanacearum inoculation (RSI) and HTHH resulted in up-regulation of this nucleus-localized TF. Function of this TF was confirmed by performing loss of function assay of CaMYB59 by VIGS (virus-induced gene silencing). Plants with silenced CaMYB59 displayed not only compromised pepper immunity against RSI but also impaired tolerance to HTHH along with decreased hypersensitive response (HR). This impairment in defense function was fully linked with low induction of stress-linked genes like CaPO2, CaPR1, CaAcc and thermo-tolerance linked CaHSP24 as well as CaHsfB2a. Conversely, transient overexpression of CaMYB59 enhanced pepper immunity. This reveals that CaMYB59 positively regulated host defense against RSI and HTHH by means of HR like mimic cell death, H2O2 production and up-regulation of defense as well as thermo-tolerance associated genes. These changes in attributes collectively confirm the role of CaMYB59 as a positive regulator of pepper immunity against R. solanacearum. We recommend that such positive regulation of pepper defense is dynamically supported by phyto-hormone signaling and transcriptional web of defense genes. These integrated and interlinked events stabilize plant growth and survival under abiotic and biotic stresses.

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.

Dynamic N6 -methyladenosine RNA modification regulates peanut resistance to bacterial wilt.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification in eukaryotes and is an important regulator of gene expression as well as many other critical biological processes. However, the characteristics and functions of m6 A in peanut (Arachis hypogea L.) resistance to bacterial wilt (BW) remain unknown. Here, we analyzed the dynamic of m6 A during infection of resistant (H108) and susceptible (H107) peanut accessions with Ralstonia solanacearum (R. solanacearum), the causative agent of BW. Throughout the transcriptome, we identified 'URUAY' as a highly conserved motif for m6 A in peanut. The majority of differential m6 A located within the 3' untranslated region (UTR) of the transcript, with fewer in the exons. Integrative analysis of RNA-Seq and m6 A methylomes suggests the correlation between m6 A and gene expression in peanut R. solanacearum infection, and functional analysis reveals that m6 A-associated genes were related to plant-pathogen interaction. Our experimental analysis suggests that AhALKBH15 is an m6 A demethylase in peanut, leading to decreased m6 A levels and upregulation of the resistance gene AhCQ2G6Y. The upregulation of AhCQ2G6Y expression appears to promote BW resistance in the H108 accession.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

In vivo application of potent probiotics for enhancing potato growth and controlling Ralstonia solanacearum and Fusarium oxysporum infections.

Plant probiotics are live microbial cells or cultures that support plant growth and control plant pathogens through different mechanisms. They have various effects on plants, including plant growth promotion through the production of indole acetic acid (IAA), biological control activity (BCA), and production of cellulase enzymes, thus inducing systemic resistance and increasing the availability of mineral elements. The present work aimed to study the potential of Achromobacter marplatensis and Bacillus velezensis as plant probiotics for the field cultivation of potatoes. In vitro studies have demonstrated the ability of selected probiotics to produce IAA and cellulase, as well as antimicrobial activity against two plant pathogens that infect Solanum tuberosum as Fusarium oxysporum and Ralstonia solanacearum under different conditions at a broad range of different temperatures and pH values. In vivo study of the effects of the probiotics A. marplatensis and B. velezensis on S. tuberosum plants grown in sandy clay loamy soil was detected after cultivation for 90 days. Probiotic isolates A. marplatensis and B. velezensis were able to tolerate ultraviolet radiation (UV) exposure for up to two hours, the dose response curve exhibited that the D10 values of A. marplatensis and B. velezensis were 28 and 16 respectively. In the case of loading both probiotics with broth, the shoot dry weight was increased significantly from 28 in the control to 50 g, shoot length increased from 24 to 45.7 cm, branches numbers increased from 40 to 70 branch, leaves number increased from 99 to 130 leaf, root dry weight increased from 9.3 to 12.9 g, root length increased from 24 to 35.7 cm, tuber weight increased from 15 to 37.0 g and tubers number increased from 9 to 24.4 tuber, the rot percentage was reduced to 0%. The addition of both probiotic isolates, either broth or wheat grains load separately has enhanced all the growth parameters; however, better results and increased production were in favor of adding probiotics with broth more than wheat. On the other hand, both probiotics showed a remarkable protective effect against potato pathogens separately and reduced the negative impact of the infection using them together.

Genome-wide analysis of the peanut CaM/CML gene family reveals that the AhCML69 gene is associated with resistance to Ralstonia solanacearum.

BACKGROUND: Calmodulins (CaMs)/CaM-like proteins (CMLs) are crucial Ca2+-binding sensors that can decode and transduce Ca2+ signals during plant development and in response to various stimuli. The CaM/CML gene family has been characterized in many plant species, but this family has not yet been characterized and analyzed in peanut, especially for its functions in response to Ralstonia solanacearum. In this study, we performed a genome-wide analysis to analyze the CaM/CML genes and their functions in resistance to R. solanacearum. RESULTS: Here, 67, 72, and 214 CaM/CML genes were identified from Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. The genes were divided into nine subgroups (Groups I-IX) with relatively conserved exon‒intron structures and motif compositions. Gene duplication, which included whole-genome duplication, tandem repeats, scattered repeats, and unconnected repeats, produced approximately 81 pairs of homologous genes in the AhCaM/CML gene family. Allopolyploidization was the main reason for the greater number of AhCaM/CML members. The nonsynonymous (Ka) versus synonymous (Ks) substitution rates (less than 1.0) suggested that all homologous pairs underwent intensive purifying selection pressure during evolution. AhCML69 was constitutively expressed in different tissues of peanut plants and was involved in the response to R. solanacearum infection. The AhCML69 protein was localized in the cytoplasm and nucleus. Transient overexpression of AhCML69 in tobacco leaves increased resistance to R. solanacearum infection and induced the expression of defense-related genes, suggesting that AhCML69 is a positive regulator of disease resistance. CONCLUSIONS: This study provides the first comprehensive analysis of the AhCaM/CML gene family and potential genetic resources for the molecular design and breeding of peanut bacterial wilt resistance.

Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum.

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms-rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

Transcriptomics and virus-induced gene silencing identify defence-related genes during Ralstonia solanacearum infection in resistant and susceptible tobacco.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a globally prevalent bacterial soil-borne disease. In this study, transcriptome sequencing were subjected to roots after infection with the R. solanacearum in the resistant and susceptible tobacco variety. DEGs that responded to R. solanacearum infection in both resistant and susceptible tobacco contributed to pectinase and peroxidase development and were enriched in plant hormone signal transduction, signal transduction and MAPK signalling pathway KEGG terms. Core DEGs in the resistant tobacco response to R. solanacearum infection were enriched in cell wall, membrane, abscisic acid and ethylene terms. qRT-PCR indicated that Nitab4.5_0004899g0110, Nitab4.5_0004234g0080 and Nitab4.5_0001439g0050 contributed to the response to R. solanacearum infection in different resistant and susceptible tobacco. Silencing the p450 gene Nitab4.5_0001439g0050 reduced tobacco resistance to bacterial wilt. These results improve our understanding of the molecular mechanism of BW resistance in tobacco and solanaceous plants.

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen.

Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

Effects of the invasion of Ralstonia solanacearum on soil microbial community structure in Wuhan, China.

This study investigated the change in the microbiome of tomato rhizosphere soils after the invasion of Ralstonia solanacearum and analyzed the correlation between microbes and soil physicochemical properties. Diversity analyses of the bacteria in healthy and diseased rhizosphere soil samples (HRS and DRS) revealed that HRS had a higher species diversity and were compositionally different from DRS (P ≤ 0.05). Substantial differences in the relative abundance of Actinobacteria (37.52% vs 28.96%, P ≤ 0.05) and Proteobacteria (29.20% vs 35.59%, P ≤ 0.05) were identified in HRS and DRS, respectively. Taxonomic composition analysis showed ten differentially abundant genera, and seven of them (Gaiella, Roseisolibacter, Solirubrobacter, Kribbella, Acidibacter, Actinomarinicola, and Marmoricola) are more abundant in HRS. Soil pH and enzyme activities were negatively correlated with the abundance of R. solanacearum. The contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkaline nitrogen (alkaline N), available phosphorus (AP), available potassium (AK), NO3-N(NN), NH4+-N (AN), and organic matter (OM) were all significantly increased in DRS. The composition and richness of protozoa in the samples show significant differences. Cephalobus, Acrobeles, Heteromita, norank_Tylenchida, and Rotylenchulus were enriched in DRS. Microbial interaction networks revealed that the HRS networks were more complex than the DRS networks. Overall, the results of this study demonstrate that healthy soil has a more complex microbial community structure and higher enzyme activity, and the invasion of R. solanacearum damages the soil microbial system.IMPORTANCEHow does the invasion of Ralstonia solanacearum affect tomato rhizosphere bacteria and protozoa? Which microbial changes can affect the growth of R. solanacearum? To date, most research studies focus on bacteria, with little research on protozoa, and even less on the synergistic effects between protozoa and bacteria. Here, we analyzed the correlation between tomato rhizosphere bacterial and protozoan communities and soil physicochemical properties during the invasion of R. solanacearum. We found that the diversity and abundance of rhizosphere microorganisms in healthy rhizosphere soil samples (HRS) were significantly higher than those in diseased rhizosphere soil samples (DRS), and there were significant changes in soil pH and enzyme activity. Overall, in this study, the analysis of microbial changes during the invasion of R. solanacearum provides a theoretical basis for the prevention and control of bacterial wilt.

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.

Ralstonia solanacearum differentially modulates soil physicochemical properties and rhizospheric bacteriome of resistant and susceptible tobacco cultivars.

Ralstonia solanacearum is a devastating soilborne pathogen which poses significant yield and economic losses to tobacco production globally. The impact of R. solanacearum on rhizosphere bacteriome and soil physicochemical characteristics in resistant and susceptible tobacco cultivars is poorly understood. This study aims to determine the effect of R. solanacearum on soil physicochemical parameters and rhizosphere bacteriome of resistant (K326) and susceptible (Hongda) tobacco cultivars at various growth stages. Results demonstrated that the contents of available potassium and phosphorus, as well as soil pH were significantly increased in K326 soils (CK and T2) compared with Hongda (T1) after 21, 42, and 63 days post-inoculation (dpi) of R. solanacearum except for available nitrogen which showed an opposite trend. The qPCR results showed a significant decrease in R. solanacearum population in rhizosphere of K326 (T2) compared to the Hongda (T1) at 21 and 63 dpi than that after 42 dpi. The rhizosphere bacteriome analysis through 16S rRNA amplicon sequencing revealed that rhizosphere bacterial community composition was significantly different between two tobacco cultivars (Hongda and K326) and this effect was more prominent after 63 dpi (93 days after post-transplantation), suggesting that each cultivar recruits a unique set of bacterial communities. There was no obvious difference observed in the rhizosphere bacteriome of CK (K326) and T2 (K326), which might be attributed to the same genetic makeup and inherent resistance of K326 to bacterial wilt infection. Analysis of co-occurrence networks revealed that the microbial network in T1 (Hongda) was more complex than those in T2 (K326) and CK (K326), while the networks in CK and T2 were almost identical. The present research highlights the time-course relationship between environmental factors and rhizosphere bacteriome of tobacco cultivars showing different levels of resistance against R. solanacearum. Conclusively, studying the plant-soil-microbe interaction system in susceptible and resistant tobacco cultivars may enable us to develop effective integrated disease control plans for the healthy production of tobacco crops.

Discovery, Identification, and Mode of Action of Phenolics from Marine-Derived Fungus Aspergillus ustus as Antibacterial Wilt Agents.

The bacterial wilt caused by Ralstonia solanacearum seriously affects crop yield and safety and is difficult to control. Biological activity-guided screening led to the isolation of 11 phenolic compounds including three undescribed compounds (carnemycin H-I and stromemycin B) from the secondary metabolites of a marine-derived Aspergillus ustus. One new compound is an unusual phenolic dimer. Their structures were elucidated by comprehensive spectroscopic data and J-based configurational analysis. The antibacterial activities of the isolated compounds against R. solanacearum were evaluated. Compound 3 exhibited excellent inhibitory activity with an MIC value of 3 µg/mL, which was comparable to that of streptomycin sulfate. Additionally, 3 significantly changed the morphology and inhibited the activity of succinate dehydrogenase (SDH) to interfere with the growth of R. solanacearum. Molecular docking was conducted to clarify the potential mechanisms of compound 3 with SDH. Further in vivo experiments demonstrated that 3 could remarkably inhibit the occurrence of bacterial wilt on tomatoes.

Recent advances in immuno-based methods for the detection of Ralstonia solanacearum.

Ralstonia solanacearum (RS) is a widely recognized phytopathogenic bacterium which is responsible for causing devastating losses in a wide range of economically significant crops. Timely and accurate detection of this pathogen is pivotal to implementing effective disease management strategies and preventing crop losses. This review provides a comprehensive overview of recent advances in immuno-based detection methods for RS. The review begins by introducing RS, highlighting its destructive potential and the need for point-of-care detection techniques. Subsequently, it explores traditional detection methods and their limitations, emphasizing the need for innovative approaches. The main focus of this review is on immuno-based detection methods and it discusses recent advancements in serological detection techniques. Furthermore, the review sheds light on the challenges and prospects of immuno-based detection of RS. It emphasizes the importance of developing rapid, field-deployable assays that can be used by farmers and researchers alike. In conclusion, this review provides valuable insights into the recent advances in immuno-based detection methods for RS.

pH-responsive bentonite nanoclay carriers control the release of benzothiazolinone to restrain bacterial wilt disease.

Ralstonia solanacearum (R. solanacearum) is one of the most devastating pathogens in terms of losses in agricultural production. Bentonite (Bent) is a promising synergistic agent used in development of effective and environmentally friendly pesticides against plant disease. However, the synergistic mechanism of Bent nanoclays with benzothiazolinone (BIT) against R. solanacearum is unknown. In this work, acid-functionalized porous Bent and cetyltrimethylammonium bromide (CTAB) were employed as the core nanoclays, and BIT was loaded into the clay to form BIT-loaded CT-Bent (BIT@CT-Bent) for the control of bacterial wilt disease. BIT@CT-Bent exhibited pH-responsive release behavior that fit the Fickian diffusion model, rapidly releasing BIT in an acidic environment (pH = 5.5). The antibacterial effect of BIT@CT-Bent was approximately 4 times greater than that of the commercial product BIT, and its biotoxicity was much lower than that of BIT under the same conditions. Interestingly, R. solanacearum attracted BIT@CT-Bent into the nanocomposites and induced cytoplasmic leakage and changes in membrane permeability, indicating an efficient and synergistic bactericidal effect that rapidly reduced bacterial density. In addition, BIT@CT-Bent significantly inhibited R. solanacearum biofilm formation and swimming activity, by suppressing the expression of phcA, solR and vsrC. Indeed, exogenous application of BIT@CT-Bent significantly suppressed the virulence of R. solanacearum on tobacco plants, with control effect of 75.48%, 72.08% and 66.08% at 9, 11 and 13 days after inoculation, respectively. This study highlights the potential of using BIT@CT-Bent as an effective, eco-friendly bactericide to control bacterial wilt diseases and for the development of sustainable crop protection strategies.

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.

Positive regulation of the PhcB neighbouring regulator PrhX on expression of the type III secretion system and pathogenesis in Ralstonia solanacearum.

Ralstonia solanacearum PhcB and PhcA control a quorum-sensing (QS) system that globally regulates expression of about one third of all genes, including pathogenesis genes. The PhcB-PhcA QS system positively regulates the production of exopolysaccharide (EPS) and negatively regulates hrp gene expression, which is crucial for the type III secretion system (T3SS). Both EPS and the T3SS are essential for pathogenicity. The gene rsc2734 is located upstream of a phcBSR operon and annotated as a response regulator of a two-component system. Here, we demonstrated that RSc2734, hereafter named PrhX, positively regulated hrp gene expression via a PrhA-PrhIR-PrhJ-HrpG signalling cascade. Moreover, PrhX was crucial for R. solanacearum to invade host roots and grow in planta naturally. prhX expression was independent of the PhcB-PhcA QS system. PrhX did not affect the expression of phcB and phcA and the QS-dependent phenotypes, such as EPS production and biofilm formation. Our results provide novel insights into the complex regulatory network of the T3SS and pathogenesis in R. solanacearum.

An ancient cis-element targeted by Ralstonia solanacearum TALE-like effectors facilitates the development of a promoter trap that could confer broad-spectrum wilt resistance.

Ralstonia solanacearum, a species complex of bacterial plant pathogens that causes bacterial wilt, comprises four phylotypes that evolved when a founder population was split during the continental drift ~180 million years ago. Each phylotype contains strains with RipTAL proteins structurally related to transcription activator-like (TAL) effectors from the bacterial pathogen Xanthomonas. RipTALs have evolved in geographically separated phylotypes and therefore differ in sequence and potentially functionality. Earlier work has shown that phylotype I RipTAL Brg11 targets a 17-nucleotide effector binding element (EBE) and transcriptionally activates the downstream arginine decarboxylase (ADC) gene. The predicted DNA binding preferences of Brg11 and RipTALs from other phylotypes are similar, suggesting that most, if not all, RipTALs target the Brg11-EBE motif and activate downstream ADC genes. Here we show that not only phylotype I RipTAL Brg11 but also RipTALs from other phylotypes activate host genes when preceded by the Brg11-EBE motif. Furthermore, we show that Brg11 and RipTALs from other phylotypes induce the same quantitative changes of ADC-dependent plant metabolites, suggesting that most, if not all, RipTALs induce functionally equivalent changes in host cells. Finally, we report transgenic tobacco lines in which the RipTAL-binding motif Brg11-EBE mediates RipTAL-dependent transcription of the executor-type resistance (R) gene Bs4C from pepper, thereby conferring resistance to RipTAL-delivering R. solanacearum strains. Our results suggest that cell death-inducing executor-type R genes, preceded by the RipTAL-binding motif Brg11-EBE, could be used to genetically engineer broad-spectrum bacterial wilt resistance in crop plants without any apparent fitness penalty.