Pathogen resistance was negatively regulated by the NAC transcription factor ScATAF1 in sugarcane.

The NAC (NAM, ATAF, and CUC) is one of the largest transcription factor gene families in plants. In this study, 180, 141, and 131 NAC family members were identified from Saccharum complex, including S. officinarum, S. spontaneum, and Erianthus rufipilus. The Ka/Ks ratio of ATAF subfamily was all less than 1. Besides, 52 ATAF members from 12 representative plants were divided into three clades and there was only a significant expansion in maize. Surprisingly, ABA and JA cis-elements were abundant in hormonal response factor, followed by transcriptional regulator and abiotic stressor. The ATAF subfamily was differentially expressed in various tissues, under low temperature and smut pathogen treatments. Further, the ScATAF1 gene, with high expression in leaves, stem epidermis, and buds, was isolated. The encoded protein, lack of self-activation activity, was situated in the cell nucleus. Moreover, SA and JA stresses down-regulated the expression of this gene, while ABA, NaCl, and 4°C treatments led to its up-regulation. Interestingly, its expression in the smut susceptible sugarcane cultivars was much higher than the smut resistant ones. Notably, the colors presented slight brown in tobacco transiently overexpressing ScATAF1 at 1 d after DAB staining, while the symptoms were more obvious at 3 d after inoculation with Ralstonia solanacearum, with ROS, JA, and SA signaling pathway genes significantly up-regulated. We thus speculated ScATAF1 gene could negatively mediate hypersensitive reactions and produce ROS by JA and SA signaling pathways. These findings lay the groundwork for in-depth investigation on the biological roles of ATAF subfamily in sugarcane.

A bacterial effector manipulates plant metabolism, cell death, and immune responses via independent mechanisms.

Bacterial pathogens inject effector proteins inside plant cells to manipulate cellular functions and achieve a successful infection. The soil-borne pathogen Ralstonia solanacearum (Smith), the causal agent of bacterial wilt disease, secretes > 70 different effectors inside plant cells, although only a handful of them have been thoroughly characterized. One of these effectors, named RipI, is required for full R. solanacearum pathogenicity. RipI associates with plant glutamate decarboxylases (GADs) to promote the accumulation of gamma-aminobutyric acid (GABA), which serves as bacterial nutrient. In this work, we found that RipI can also suppress plant immune responses to bacterial elicitors, which seems to be unrelated to the ability of RipI to induce GABA accumulation and plant cell death. A detailed characterization of the RipI features that contribute to its virulence activities identified two residues at the C-terminal domain that mediate RipI interaction with plant GADs and the subsequent promotion of GABA accumulation. These residues are also required for the appropriate homeostasis of RipI in plant cells and the induction of cell death, although they are partially dispensable for the suppression of plant immune responses. Altogether, we decipher and uncouple the virulence activities of an important bacterial effector at the biochemical level.

Sugarcane ScOPR1 gene enhances plant disease resistance through the modulation of hormonal signaling pathways.

KEY MESSAGE: Transgenic plants stably overexpressing ScOPR1 gene enhanced disease resistance by increasing the accumulation of JA, SA, and GST, as well as up-regulating the expression of genes related to signaling pathways. 12-Oxo-phytodienoate reductase (OPR) is an oxidoreductase that depends on flavin mononucleotide (FMN) and catalyzes the conversion of 12-oxophytodienoate (12-OPDA) into jasmonic acid (JA). It plays a key role in plant growth and development, and resistance to adverse stresses. In our previous study, we have obtained an OPR gene (ScOPR1, GenBank Accession Number: MG755745) from sugarcane. This gene showed positive responses to methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), and Sporisorium scitamineum, suggesting its potential for pathogen resistance. Here, in our study, we observed that Nicotiana benthamiana leaves transiently overexpressing ScOPR1 exhibited weaker disease symptoms, darker 3,3-diaminobenzidine (DAB) staining, higher accumulation of reactive oxygen species (ROS), and higher expression of hypersensitive response (HR) and SA pathway-related genes after inoculation with Ralstonia solanacearum and Fusarium solanacearum var. coeruleum. Furthermore, the transgenic N. benthamiana plants stably overexpressing the ScOPR1 gene showed enhanced resistance to pathogen infection by increasing the accumulation of JA, SA, and glutathione S-transferase (GST), as well as up-regulating genes related to HR, JA, SA, and ROS signaling pathways. Transcriptome analysis revealed that the specific differentially expressed genes (DEGs) in ScOPR1-OE were significantly enriched in hormone transduction signaling and plant-pathogen interaction pathways. Finally, a functional mechanism model of the ScOPR1 gene in response to pathogen infection was depicted. This study provides insights into the molecular mechanism of ScOPR1 and presents compelling evidence supporting its positive involvement in enhancing plant disease resistance.

Pepper immunity against Ralstonia solanacearum is positively regulated by CaWRKY3 through modulation of different WRKY transcription factors.

BACKGROUND: Several WRKY transcription factors (TFs), including CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 are known to govern the resistance of pepper (Capsicum annuum L.) plants to Ralstonia solanacearum infestation (RSI) and other abiotic stresses. However, the molecular mechanisms underlying these processes remain elusive. METHODS: This study functionally described CaWRKY3 for its role in pepper immunity against RSI. The roles of phytohormones in mediating the expression levels of CaWRKY3 were investigated by subjecting pepper plants to 1 mM salicylic acid (SA), 100 µM methyl jasmonate (MeJA), and 100 µM ethylene (ETH) at 4-leaf stage. A virus-induced gene silencing (VIGS) approach based on the Tobacco Rattle Virus (TRV) was used to silence CaWRKY3 in pepper, and transiently over-expressed to infer its role against RSI. RESULTS: Phytohormones and RSI increased CaWRKY3 transcription. The transcriptions of defense-associated marker genes, including CaNPR1, CaPR1, CaDEF1, and CaHIR1 were decreased in VIGS experiment, which made pepper less resistant to RSI. Significant hypersensitive (HR)-like cell death, H2O2 buildup, and transcriptional up-regulation of immunological marker genes were noticed in pepper when CaWRKY3 was transiently overexpressed. Transcriptional activity of CaWRKY3 was increased with overexpression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40, and vice versa. In contrast, Pseudomonas syringae pv tomato DC3000 (Pst DC3000) was easily repelled by the innate immune system of transgenic Arabidopsis thaliana that overexpressed CaWRKY3. The transcriptions of defense-related marker genes like AtPR1, AtPR2, and AtNPR1 were increased in CaWRKY3-overexpressing transgenic A. thaliana plants. CONCLUSION: It is concluded that CaWRKY3 favorably regulates phytohormone-mediated synergistic signaling, which controls cell death in plant and immunity of pepper plant against bacterial infections.

Naringenin restricts the colonization and growth of Ralstonia solanacearum in tobacco mutant KCB-1.

Bacterial wilt severely jeopardizes plant growth and causes enormous economic loss in the production of many crops, including tobacco (Nicotiana tabacum). Here, we first demonstrated that the roots of bacterial wilt-resistant tobacco mutant KCB-1 can limit the growth and reproduction of Ralstonia solanacearum. Secondly, we demonstrated that KCB-1 specifically induced an upregulation of naringenin content in root metabolites and root secretions. Further experiments showed that naringenin can disrupt the structure of R. solanacearum, inhibit the growth and reproduction of R. solanacearum, and exert a controlling effect on bacterial wilt. Exogenous naringenin application activated the resistance response in tobacco by inducing the burst of reactive oxygen species and salicylic acid deposition, leading to transcriptional reprogramming in tobacco roots. Additionally, both external application of naringenin in CB-1 and overexpression of the Nicotiana tabacum chalcone isomerase (NtCHI) gene, which regulates naringenin biosynthesis, in CB-1 resulted in a higher complexity of their inter-root bacterial communities than in untreated CB-1. Further analysis showed that naringenin could be used as a marker for resistant tobacco. The present study provides a reference for analyzing the resistance mechanism of bacterial wilt-resistant tobacco and controlling tobacco bacterial wilt.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Genetic Diversity of Ralstonia solanacearum Causing Tobacco Bacterial Wilt in Fujian Province and Identification of Biocontrol Streptomyces sp.

Tobacco bacterial wilt is a highly destructive soilborne disease caused by the Ralstonia solanacearum species complex, exhibiting a significant risk to global flue-cured tobacco cultivation and resulting in substantial economic loss. In this study, 77 isolates were collected from three prominent flue-cured tobacco cultivation areas in Fujian, China (Nanping, Sanming, and Longyan), in 2021 and 2022. The isolated strains were classified through phylotype-specific multiplex polymerase chain reaction (Pmx-PCR) and physiological tests. The analysis showed that all the strains were associated with phylotype I, race 1, and biovar III. Subsequent phylogenetic analysis using partial egl gene sequences classified the 77 isolates into 5 distinct sequevars: 13, 15, 16, 17, and 34. Notably, a remarkable predominance of sequevar 15 was observed in Fujian Province, while sequevar 16 was first reported on tobacco in China, which was identified in other plants, expanding the understanding of its host range and distribution in the country. In addition, a Streptomyces strain extracted from the rhizosphere soil of tobacco was found to inhibit the growth of multiple sequevars of tobacco R. solanacearum, indicating its broad-spectrum antagonistic properties. Furthermore, pot experiments showed that the strain St35 effectively controlled tobacco bacterial wilt. The isolate St35 was conclusively identified as Streptomyces gancidicus according to the morphological and genetic features. In summary, the present study demonstrated the genetic diversity and distribution of tobacco R. solanacearum strains in the Fujian province of China, as well as the identification of a candidate biological control agent for the management of tobacco bacterial wilt.

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.

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.

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.

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.

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.

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.

Digital gene expression analysis of the response to Ralstonia solanacearum between resistant and susceptible tobacco varieties.

Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious soil-borne disease of tobacco. However, molecular mechanism information of R. solanacearum resistance is limited to tobacco, hindering better breeding of resistant tobacco. In this study, the expression profiles of the rootstalks of Yunyan87 (susceptible cultivar) and Fandi3 (resistant cultivar) at different stages after R. solanacearum infection were compared to explore molecular mechanisms of tobacco resistance against the bacterium. Findings from gene-expression profiling indicated that the number of upregulated differentially expressed genes (DEGs) at 3 and 7 days post-inoculation (dpi) increased significantly in the resistant cultivar. WRKY6 and WRKY11 family genes in WRKY transcription factors, ERF5 and ERF15 family genes in ERFs transcription factors, and genes encoding PR5 were significantly upregulated in the resistant cultivar response to the infection. For the first time, WRKY11 and ERF15 were found to be possibly involved in disease-resistance. The Kyoto Encyclopedia of Genes and Genomes analysis demonstrated glutathione metabolism and phenylpropane pathways as primary resistance pathways to R. solanacearum infection. In the resistant cultivar, DEGs encoding CYP450, TCM, CCoAOMT, 4CL, PAL, CCR, CSE, and CADH, involved in the synthesis of plant antitoxins such as flavonoids, stilbenoids, and lignins, enriched in the phenylpropane pathway were upregulated at 3 and 7 dpi. Furthermore, a pot experiment was performed to verify the role of flavonoids in controlling TBW. This study will strongly contribute to a better understanding of molecular interactions between tobacco plants and R. solanacearum.

Characterization of the mechanism of thioredoxin-dependent activation of γ-glutamylcyclotransferase, RipAY, from Ralstonia solanacearum.

A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 1:2 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.