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.

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.

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.

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.

Ralstonia solanacearum pandemic lineage strain UW551 overcomes inhibitory xylem chemistry to break tomato bacterial wilt resistance.

Plant-pathogenic Ralstonia strains cause bacterial wilt disease by colonizing xylem vessels of many crops, including tomato. Host resistance is the best control for bacterial wilt, but resistance mechanisms of the widely used Hawaii 7996 tomato breeding line (H7996) are unknown. Using growth in ex vivo xylem sap as a proxy for host xylem, we found that Ralstonia strain GMI1000 grows in sap from both healthy plants and Ralstonia-infected susceptible plants. However, sap from Ralstonia-infected H7996 plants inhibited Ralstonia growth, suggesting that in response to Ralstonia infection, resistant plants increase inhibitors in their xylem sap. Consistent with this, reciprocal grafting and defence gene expression experiments indicated that H7996 wilt resistance acts in both above- and belowground plant parts. Concerningly, H7996 resistance is broken by Ralstonia strain UW551 of the pandemic lineage that threatens highland tropical agriculture. Unlike other Ralstonia, UW551 grew well in sap from Ralstonia-infected H7996 plants. Moreover, other Ralstonia strains could grow in sap from H7996 plants previously infected by UW551. Thus, UW551 overcomes H7996 resistance in part by detoxifying inhibitors in xylem sap. Testing a panel of xylem sap compounds identified by metabolomics revealed that no single chemical differentially inhibits Ralstonia strains that cannot infect H7996. However, sap from Ralstonia-infected H7996 contained more phenolic compounds, which are known to be involved in plant antimicrobial defence. Culturing UW551 in this sap reduced total phenolic levels, indicating that the resistance-breaking Ralstonia strain degrades these chemical defences. Together, these results suggest that H7996 tomato wilt resistance depends in part on inducible phenolic compounds in xylem sap.

Transcriptomic profiling reveals host-specific evolutionary pathways promoting enhanced fitness in the plant pathogen Ralstonia pseudosolanacearum.

The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain.

IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

Transcriptomic analysis of Ralstonia solanacearum in response to antibacterial volatiles of Bacillus velezensis FZB42.

Volatile organic compounds (VOCs), produced by a variety of microbial species and used as biological agents, have been demonstrated to play a significant role in controlling phytopathogens. In continuation of our previous studies, we aim to elucidate the underlying mechanisms and pathways involved in interactions between pathogens and microbial VOCs. In the current study, we tested how VOCs produced by Bacillus velezensis FZB42 affect the growth of Ralstonia solanacearum TBBS1 in vitro.Query The result showed that the colony growth of R. solanacearum was reduced with an inhibition rate of 0.83 ± 0.043 as compared to the control 1.7 ± 0.076, respectively. The number of viable cells of R. solanacearum was significantly decreased to 7.68 CFU/mL as compared to the control (9.02 CFU/mL). In addition, transcriptomic analysis of R. solanacearum in response to VOCs produced by FZB42 was performed to better understand the effect of VOCs on R. solanacearum. The transcriptional response of R. solanacearum to FZB42-VOCs was determined using an Illumina RNA-seq approach. The results revealed significant changes in the expression of 2094 R. solanacearum genes, including 593 upregulated and 1501 downregulated genes. To validate the RNA-seq results, the expression of 10 genes was quantified using RT-qPCR. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to functionally annotate differentially expressed genes. Significant changes were observed in genes directly or indirectly related to virulence, including those related to bacterial invasion, motility, chemotaxis, and secretion systems. Overall, RNA-seq profiling provides new insights into the possible fundamental molecular mechanisms that are responsible for the reduction in growth and virulence of R. solanacearum upon application of FZB42-VOC.

Integrative transcriptomic analysis unveils lncRNA-miRNA-mRNA interplay in tomato plants responding to Ralstonia solanacearum.

Ralstonia solanacearum, a bacterial plant pathogen, poses a significant threat to tomato (Solanum lycopersicum) production through destructive wilt disease. While noncoding RNA has emerged as a crucial regulator in plant disease, its specific involvement in tomato bacterial wilt remains limited. Here, we conducted a comprehensive analysis of the transcriptional landscape, encompassing both mRNAs and noncoding RNAs, in a tomato resistant line ('ZRS_7') and a susceptible line ('HTY_9') upon R. solanacearum inoculation using high-throughput RNA sequencing. Differential expression (DE) analysis revealed significant alterations in 7506 mRNAs, 997 lncRNAs, and 69 miRNAs between 'ZRS_7' and 'HTY_9' after pathogen exposure. Notably, 4548 mRNAs, 367 lncRNAs, and 26 miRNAs exhibited genotype-specific responses to R. solanacearum inoculation. GO and KEGG pathway analyses unveiled the potential involvement of noncoding RNAs in the response to bacterial wilt disease, targeting receptor-like kinases, cell wall-related genes, glutamate decarboxylases, and other key pathways. Furthermore, we constructed a comprehensive competing endogenous RNA (ceRNA) network incorporating 13 DE-miRNAs, 30 DE-lncRNAs, and 127 DEGs, providing insights into their potential contributions to the response against bacterial inoculation. Importantly, the characterization of possible endogenous target mimics (eTMs) of Sly-miR482e-3p via VIGS technology demonstrated the significant impact of eTM482e-3p-1 silencing on tomato's sensitivity to R. solanacearum. These findings support the existence of an eTM482e-3p-1-Sly-miR482e-3p-NBS-LRRs network in regulating tomato's response to the pathogen. Collectively, our findings shed light on the intricate interactions among lncRNAs, miRNAs, and mRNAs as underlying factors in conferring resistance to R. solanacearum in tomato.

Part II: Impedance-based DNA biosensor for detection of isolated strains of phytopathogen Ralstonia solanacearum.

In Part I, we demonstrated the complete development of a label-free, ultra-low sample volume requiring DNA-based biosensor to detect Ralstonia solanacearum, an aerobic non-spore-forming, Gram-negative, plant pathogenic bacterium, using non-faradaic electrochemical impedance spectroscopy (nf-EIS). We also presented the sensor's sensitivity, specificity, and electrochemical stability. In this article, we highlight the specificity study of the developed DNA-based impedimetric biosensor to detect various strains of R. solanacearum. We have collected seven isolates of R. solanacearum isolated from locally infected host plants (eggplant, potato, tomato, chilli, and ginger) from different parts of Goa, India. The pathogenicity of these isolates was tested on the eggplant, and the pathogen was confirmed by microbiological plating and polymerase chain reaction (PCR). We further report the insight into the DNA hybridization on the surface of Interdigitated Electrodes (IDEs) and the expansion of the Randles model for more accurate analysis. The interpretation of the sensor specificity is clearly demonstrated by the capacitance change observed at the electrode-electrolyte interface.

Insights into the metabolic specificities of pathogenic strains from the Ralstonia solanacearum species complex.

All the strains grouped under the species Ralstonia solanacearum represent a species complex responsible for many diseases on agricultural crops throughout the world. The strains have different lifestyles and host range. Here, we investigated whether specific metabolic pathways contribute to strain diversification. To this end, we carried out systematic comparisons on 11 strains representing the diversity of the species complex. We reconstructed the metabolic network of each strain from its genome sequence and looked for the metabolic pathways differentiating the different reconstructed networks and, by extension, the different strains. Finally, we conducted an experimental validation by determining the metabolic profile of each strain with the Biolog technology. Results revealed that the metabolism is conserved between strains, with a core metabolism composed of 82% of the pan-reactome. The three species composing the species complex could be distinguished according to the presence/absence of some metabolic pathways, in particular, one involving salicylic acid degradation. Phenotypic assays revealed that the trophic preferences on organic acids and several amino acids such as glutamine, glutamate, aspartate, and asparagine are conserved between strains. Finally, we generated mutants lacking the quorum-sensing-dependent regulator PhcA in four diverse strains, and we showed that the phcA-dependent trade-off between growth and production of virulence factors is conserved across the R. solanacearum species complex. IMPORTANCE Ralstonia solanacearum is one of the most important threats to plant health worldwide, causing disease on a very large range of agricultural crops such as tomato or potato. Behind the R. solanacearum name are hundreds of strains with different host range and lifestyle, classified into three species. Studying the differences between strains allows to better apprehend the biology of the pathogens and the specificity of some strains. None of the published genomic comparative studies have focused on the metabolism of the strains so far. We developed a new bioinformatic pipeline to build high-quality metabolic networks and used a combination of metabolic modeling and high-throughput phenotypic Biolog microplates to look for the metabolic differences between 11 strains across the three species. Our study revealed that genes encoding enzymes are overall conserved, with few variations between strains. However, more variations were observed when considering substrate usage. These variations probably result from regulation rather than the presence or absence of enzymes in the genome.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.

The phc Quorum-Sensing System in Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex (RSSC) strains are devastating plant pathogens distributed worldwide. The primary cell density-dependent gene expression system in RSSC strains is phc quorum sensing (QS). It regulates the expression of about 30% of all genes, including those related to cellular activity, primary and secondary metabolism, pathogenicity, and more. The phc regulatory elements encoded by the phcBSRQ operon and phcA gene play vital roles. RSSC strains use methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the QS signal. Each type of RSSC strain has specificity in generating and receiving its QS signal, but their signaling pathways might not differ significantly. In this review, I describe the genetic and biochemical factors involved in QS signal input and the regulatory network and summarize control of the phc QS system, new cell-cell communications, and QS-dependent interactions with soil fungi.

Complete genome sequencing of Bacillus subtilis (CWTS 5), a siderophore-producing bacterium triggers antagonistic potential against Ralstonia solanacearum.

AIM: The aims of this study were to explore the antagonistic potential of siderophore-producing Bacillus subtilis (CWTS 5) for the suppression of Ralstonia solanacearum and to explore the mechanisms of inhibition by FTIR, LC-MS, and whole genome analysis. METHODS AND RESULTS: A siderophore-producing B. subtilis (CWTS 5) possessing several plant growth-promoting properties such as IAA and ACC deaminase production, phosphate solubilization, and nitrogen fixation was assessed for its inhibitory effect against R. solanacearum, and its mechanisms were explored by in vitro and in vivo analyses. The active secondary metabolites in the siderophore extracts were identified as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone by LC-MS analysis. The Arnow's test and antiSMASH analysis confirmed the presence of catecholate siderophores, and the functional groups determined by FTIR spectroscopy confirmed the presence of secondary metabolites in the siderophore extract possessing antagonistic effect. The complete genome sequence of CWTS 5 revealed the gene clusters responsible for siderophore, antibiotics, secondary metabolite production, and antibacterial and antifungal metabolites. Furthermore, the evaluation of CWTS 5 against R. solanacearum in pot studies demonstrated 40.0% reduced disease severity index (DSI) by CWTS 5, methanolic extract (DSI-26.6%), ethyl acetate extract (DSI-20.0%), and increased plant growth such as root and shoot length, wet weight and dry weight of Solanum lycopersicum L. owing to its antagonistic potential. This genomic insight will support future studies on the application of B. subtilis as a plant growth promoter and biocontrol agent against R. solanacearum for bacterial wilt management. CONCLUSION: The results of this study revealed that B. subtilis (CWTS 5) possesses multiple mechanisms that control R. solanacearum, reduce disease incidence, and improve S. lycopersicum growth.

ETI signaling nodes are involved in resistance of Hawaii 7996 to Ralstonia solanacearum-induced bacterial wilt disease in tomato.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is a destructive disease of tomato. Tomato cultivar Hawaii 7996 is well-known for its stable resistance against R. solanacearum. However, the resistance mechanism of Hawaii 7996 has not yet been revealed. Here, we showed that Hawaii 7996 activated root cell death response and exhibited stronger defense gene induction than the susceptible cultivar Moneymaker after R. solanacearum GMI1000 infection. By employing virus-induced gene silencing (VIGS) and CRISPR/Cas9 technologies, we found that SlNRG1-silenced and SlADR1-silenced/knockout mutant tomato partially or completely lost resistance to bacterial wilt, indicating that helper NLRs SlADR1 and SlNRG1, the key nodes of effector-triggered immunity (ETI) pathways, are required for Hawaii 7996 resistance. In addition, while SlNDR1 was dispensable for the resistance of Hawaii 7996 to R. solanacearum, SlEDS1, SlSAG101a/b, and SlPAD4 were essential for the immune signaling pathways in Hawaii 7996. Overall, our results suggested that robust resistance of Hawaii 7996 to R. solanacearum relied on the involvement of multiple conserved key nodes of the ETI signaling pathways. This study sheds light on the molecular mechanisms underlying tomato resistance to R. solanacearum and will accelerate the breeding of tomatoes resilient to diseases.

Gene enrichment and co-expression analysis shed light on transcriptional responses to Ralstonia solanacearum in tomato.

BACKGROUND: Tomato (Solanum lycopersicum) is both an important agricultural product and an excellent model system for studying plant-pathogen interactions. It is susceptible to bacterial wilt caused by Ralstonia solanacearum (Rs), and infection can result in severe yield and quality losses. To investigate which genes are involved in the resistance response to this pathogen, we sequenced the transcriptomes of both resistant and susceptible tomato inbred lines before and after Rs inoculation. RESULTS: In total, 75.02 Gb of high-quality reads were generated from 12 RNA-seq libraries. A total of 1,312 differentially expressed genes (DEGs) were identified, including 693 up-regulated and 621 down-regulated genes. Additionally, 836 unique DEGs were obtained when comparing two tomato lines, including 27 co-expression hub genes. A total of 1,290 DEGs were functionally annotated using eight databases, most of which were found to be involved in biological pathways such as DNA and chromatin activity, plant-pathogen interaction, plant hormone signal transduction, secondary metabolite biosynthesis, and defense response. Among the core-enriched genes in 12 key pathways related to resistance, 36 genotype-specific DEGs were identified. RT-qPCR integrated analysis revealed that multiple DEGs may play a significant role in tomato response to Rs. In particular, Solyc01g073985.1 (NLR disease resistance protein) and Solyc04g058170.1 (calcium-binding protein) in plant-pathogen interaction are likely to be involved in the resistance. CONCLUSION: We analyzed the transcriptomes of both resistant and susceptible tomato lines during control and inoculated conditions and identified several key genotype-specific hub genes involved in a variety of different biological processes. These findings lay a foundation for better understanding the molecular basis by which resistant tomato lines respond to Rs.