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Cytokinin signalling plays both positive and negative roles in plant resistance to pathogens. It is not clear whether the role of cytokinin changes at the different stages of pathogen infection. Arabidopsis thaliana sequentially exhibits distinct root morphological symptoms during Ralstonia solanacearum infection, which offers a good system to investigate function of cytokinin in the whole pathogen infection process. Using this system, we found increase of cytokinin signalling by Lonely Guy 2 (LOG2) overexpression or depletion of type-A Arabidopsis Response Regulators (ARRs), negative regulators of cytokinin signalling pathway, promoted cell death, wilting symptom and bacterial growth, but attenuated primary root growth inhibition and lateral root formation. The decrease of cytokinin signalling by mutation on Isopentenyl Transferases (IPTs) inhibited root hair formation, cell death, wilting symptom and bacterial colonisation. Application of different concentration of exogenesis 6-benzylaminopurine (6-BA) showed first promoted, then decreased root hair formation. Moreover, application of 6-BA accelerated cell death but suppressed lateral root formation and primary root growth inhibition. The diverse roles of cytokinin in these different root disease phenotypes suggested function of cytokinin during plant responses to R. solanacearum is cell type-specific, which provides new insights on roles of cytokinin signalling in regulation on plant-pathogen interactions., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity. We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression. To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis. This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 de Pedro-Jové et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N 1-107 ) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N 1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant-pathogen co-evolution., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host's root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Ralstonia solanacearum is the causal agent of bacterial wilt, a devastating plant disease responsible for serious economic losses especially on potato, tomato, and other solanaceous plant species in temperate countries. In R. solanacearum, gene expression analysis has been key to unravel many virulence determinants as well as their regulatory networks. However, most of these assays have been performed using either bacteria grown in minimal medium or in planta, after symptom onset, which occurs at late stages of colonization. Thus, little is known about the genetic program that coordinates virulence gene expression and metabolic adaptation along the different stages of plant infection by R. solanacearum., Results: We performed an RNA-sequencing analysis of the transcriptome of bacteria recovered from potato apoplast and from the xylem of asymptomatic or wilted potato plants, which correspond to three different conditions (Apoplast, Early and Late xylem). Our results show dynamic expression of metabolism-controlling genes and virulence factors during parasitic growth inside the plant. Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. Xylem-induced genes included virulence genes, such as the type III secretion system (T3SS) and most of its related effectors and nitrogen utilisation genes. The upstream regulators of the T3SS were exclusively up-regulated in the apoplast, preceding the induction of their downstream targets. Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages., Conclusions: This is the first report describing R. solanacearum dynamic transcriptional changes within the plant during infection. Our data define four main genetic programmes that define gene pathogen physiology during plant colonisation. The described expression of virulence genes, which might reflect bacterial states in different infection stages, provides key information on the R. solanacearum potato infection process.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Ralstonia solanacearum is a bacterial vascular pathogen causing devastating bacterial wilt. In the field, resistance against this pathogen is quantitative and is available for breeders only in tomato and eggplant. To understand the basis of resistance to R. solanacearum in tomato, we investigated the spatio-temporal dynamics of bacterial colonization using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We found four 'bottlenecks' that limit the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion, and radial apoplastic spread in the cortex. Radial invasion of cortical extracellular spaces occurred mostly at late disease stages but was observed throughout plant infection. This study shows that resistance is expressed in both root and shoot tissues, and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads out of the xylem, occupying the plant apoplast niche. Our work will help elucidate the complex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new precision tools to fight bacterial wilt in the field., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Ralstonia solanacearum is a bacterial plant pathogen causing important economic losses worldwide. In addition to the polar flagella responsible for swimming motility, this pathogen produces type IV pili (TFP) that govern twitching motility, a flagellum-independent movement on solid surfaces. The implication of chemotaxis in plant colonization, through the control flagellar rotation by the proteins CheW and CheA, has been previously reported in R. solanacearum In this work, we have identified in this bacterium homologues of the Pseudomonas aeruginosa pilI and chpA genes, suggested to play roles in TFP-associated motility analogous to those played by the cheW and cheA genes, respectively. We demonstrate that R. solanacearum strains with a deletion of the pilI or the chpA coding region show normal swimming and chemotaxis but altered biofilm formation and reduced twitching motility, transformation efficiency, and root attachment. Furthermore, these mutants displayed wild-type growth in planta and impaired virulence on tomato plants after soil-drench inoculations but not when directly applied to the xylem. Comparison with deletion mutants for pilA and fliC -encoding the major pilin and flagellin subunits, respectively-showed that both twitching and swimming are required for plant colonization and full virulence. This work proves for the first time the functionality of a pilus-mediated pathway encoded by pil - chp genes in R. solanacearum , demonstrating that pilI and chpA genes are bona fide motility regulators controlling twitching motility and its three related phenotypes: virulence, natural transformation, and biofilm formation. IMPORTANCE Twitching and swimming are two bacterial movements governed by pili and flagella. The present work identifies for the first time in the Gram-negative plant pathogen Ralstonia solanacearum a pilus-mediated chemotaxis pathway analogous to that governing flagellum-mediated chemotaxis. We show that regulatory genes in this pathway control all of the phenotypes related to pili, including twitching motility, natural transformation, and biofilm formation, and are also directly implicated in virulence, mainly during the first steps of the plant infection. Our results show that pili have a higher impact than flagella on the interaction of R. solanacearum with tomato plants and reveal new types of cross-talk between the swimming and twitching motility phenotypes: enhanced swimming in bacteria lacking pili and a role for the flagellum in root attachment., (Copyright © 2020 Corral et al.)

Bacterial wilt caused by the bacterial pathogen Ralstonia solanacearum is one of the most devastating crop diseases worldwide. The molecular mechanisms controlling the early stage of R. solanacearum colonization in the root remain unknown. Aiming to better understand the mechanism of the establishment of R. solanacearum infection in root, we established four stages in the early interaction of the pathogen with Arabidopsis roots and determined the transcriptional profiles of these stages of infection. A total 2,698 genes were identified as differentially expressed genes during the initial 96 h after infection, with the majority of changes in gene expression occurring after pathogen-triggered root-hair development observed. Further analysis of differentially expressed genes indicated sequential activation of multiple hormone signaling cascades, including abscisic acid (ABA), auxin, jasmonic acid, and ethylene. Simultaneous impairment of ABA receptor genes promoted plant wilting symptoms after R. solanacearum infection but did not affect primary root growth inhibition or root-hair and lateral root formation caused by R. solanacearum . This indicated that ABA signaling positively regulates root defense to R. solanacearum . Moreover, transcriptional changes of genes involved in primary root, lateral root, and root-hair formation exhibited high temporal dynamics upon infection. Taken together, our results suggest that successful infection of R. solanacearum on roots is a highly programmed process involving in hormone crosstalk.

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds-including molecules effective against human pathogens-for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down-regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases., (© 2018 BSPP and John Wiley & Sons Ltd.)

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum , the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum , most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The causal agent of bacterial wilt, Ralstonia solanacearum, is a soilborne pathogen that invades plants through their roots, traversing many tissue layers until it reaches the xylem, where it multiplies and causes plant collapse. The effects of R. solanacearum infection are devastating, and no effective approach to fight the disease is so far available. The early steps of infection, essential for colonization, as well as the early plant defense responses remain mostly unknown. Here, we have set up a simple, in vitro Arabidopsis thaliana-R. solanacearum pathosystem that has allowed us to identify three clear root phenotypes specifically associated to the early stages of infection: root-growth inhibition, root-hair formation, and root-tip cell death. Using this method, we have been able to differentiate, on Arabidopsis plants, the phenotypes caused by mutants in the key bacterial virulence regulators hrpB and hrpG, which remained indistinguishable using the classical soil-drench inoculation pathogenicity assays. In addition, we have revealed the previously unknown involvement of auxins in the root rearrangements caused by R. solanacearum infection. Our system provides an easy-to-use, high-throughput tool to study R. solanacearum aggressiveness. Furthermore, the observed phenotypes may allow the identification of bacterial virulence determinants and could even be used to screen for novel forms of early plant resistance to bacterial wilt.

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.