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.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

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.

Walking down the phosphorylation path to root immunity.

Pathogen perception in plant roots is under-explored compared to that in shoots. In this issue of Cell Host & Microbe, Wang et al. characterize the phosphorylation-mediated signaling pathway that positively and negatively regulates plant resistance to bacterial wilt disease upon perception of a metabolite from the soil-borne vascular pathogen Ralstonia solanacearum.

Inoculation of Arabidopsis seedlings with Ralstonia solanacearum in sterile agar plates.

Ralstonia solanacearum invades plants through their roots and causes devastating bacterial diseases in multiple crops. Here, we present a versatile inoculation assay in Arabidopsis thaliana seedlings grown in sterile agar plates. We describe steps for plant preparation, bacterial inoculation, tissue sampling, and bacterial quantification. This protocol can be used for accurate assessment of bacterial colonization and observation of plant response to infection. For complete details on the use and execution of this protocol, please refer to Dindas et al. (2022).1.

Phytophthora sojae boosts host trehalose accumulation to acquire carbon and initiate infection.

Successful infection by pathogenic microbes requires effective acquisition of nutrients from their hosts. Root and stem rot caused by Phytophthora sojae is one of the most important diseases of soybean (Glycine max). However, the specific form and regulatory mechanisms of carbon acquired by P. sojae during infection remain unknown. In the present study, we show that P. sojae boosts trehalose biosynthesis in soybean through the virulence activity of an effector PsAvh413. PsAvh413 interacts with soybean trehalose-6-phosphate synthase 6 (GmTPS6) and increases its enzymatic activity to promote trehalose accumulation. P. sojae directly acquires trehalose from the host and exploits it as a carbon source to support primary infection and development in plant tissue. Importantly, GmTPS6 overexpression promoted P. sojae infection, whereas its knockdown inhibited the disease, suggesting that trehalose biosynthesis is a susceptibility factor that can be engineered to manage root and stem rot in soybean.

A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

A plasma membrane nucleotide-binding leucine-rich repeat receptor mediates the recognition of the Ralstonia pseudosolanacearum effector RipY in Nicotiana benthamiana.

Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops. Only a few functional resistance genes against bacterial wilt have been cloned to date. Here, we show that the broadly conserved type III secreted effector RipY is recognized by the Nicotiana benthamiana immune system, leading to cell death induction, induction of defense-related gene expression, and restriction of bacterial pathogen growth. Using a multiplexed virus-induced gene-silencing-based N. benthamiana nucleotide-binding and leucine-rich repeat receptor (NbNLR) library, we identified a coiled-coil (CC) nucleotide-binding and leucine-rich repeat receptor (CNL) required for recognition of RipY, which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY (RRS-Y). Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death and RipY-induced immunity to Ralstonia pseudosolanacearum. RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1, ACTIVATED DISEASE RESISTANCE 1, and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2, -3, and -4 in N. benthamiana. We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition. RRS-Y also broadly recognizes RipY homologs across Ralstonia species. Lastly, we show that the C-terminal region of RipY is indispensable for RRS-Y activation. Together, our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.

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.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis.

Quantitative disease resistance (QDR) remains the most prevalent form of plant resistance in crop fields and wild habitats. Genome-wide association studies (GWAS) have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR. To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum, we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R. solanacearum type III effector (T3E) mutants, identified as key pathogenicity determinants after a first screen on an A. thaliana core collection of 25 accessions. Although most quantitative trait loci (QTLs) were highly specific to the identity of the T3E mutant (ripAC, ripAG, ripAQ, and ripU), we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat (NLR) genes that exhibited structural variation. We functionally validated one of these NLRs as a susceptibility factor in response to R. solanacearum, named it Bacterial Wilt Susceptibility 1 (BWS1), and cloned two alleles that conferred contrasting levels of QDR. Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R. solanacearum effectors. In addition, we showed a direct interaction between BWS1 and RipAC T3E, and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b), the latter interaction being suppressed by RipAC. Together, our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC, mediating negative regulation of the SGT1-dependent immune response.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

Manipulation of plant metabolism by pathogen effectors: more than just food.

To successfully infect plants, pathogens secrete effector proteins to the plant apoplast or inside plant cells, where they suppress plant immunity or interfere with other cellular processes to facilitate infection. Plant metabolism is crucial for most cellular processes and plays a key role in defense against pathogens, making it a major target for pathogen effectors. Effector proteins manipulate host metabolism to provide the pathogen with nutrients or to indirectly suppress plant chemical defense responses. Recent studies have shown that pathogens also utilize effectors to shape the microbiota composition by altering the concentration of certain plant metabolites. Here, we summarize current knowledge on the manipulation of plant metabolism by pathogen effectors. We also discuss what remains unknown regarding the manipulation of host metabolism by pathogen effectors.

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

Dysfunction of histone demethylase IBM1 in Arabidopsis causes autoimmunity and reshapes the root microbiome.

Root microbiota is important for plant growth and fitness. Little is known about whether and how the assembly of root microbiota may be controlled by epigenetic regulation, which is crucial for gene transcription and genome stability. Here we show that dysfunction of the histone demethylase IBM1 (INCREASE IN BONSAI METHYLATION 1) in Arabidopsis thaliana substantially reshaped the root microbiota, with the majority of the significant amplicon sequence variants (ASVs) being decreased. Transcriptome analyses of plants grown in soil and in sterile growth medium jointly disclosed salicylic acid (SA)-mediated autoimmunity and production of the defense metabolite camalexin in the ibm1 mutants. Analyses of genome-wide histone modifications and DNA methylation highlighted epigenetic modifications permissive for transcription at several important defense regulators. Consistently, ibm1 mutants showed increased resistance to the pathogen Pseudomonas syringae DC3000 with stronger immune responses. In addition, ibm1 showed substantially impaired plant growth promotion in response to beneficial bacteria; the impairment was partially mimicked by exogenous application of SA to wild-type plants, and by a null mutation of AGP19 that is important for cell expansion and that is repressed with DNA hypermethylation in ibm1. IBM1-dependent epigenetic regulation imposes strong and broad impacts on plant-microbe interactions and thereby shapes the assembly of root microbiota.

Direct inhibition of phosphate transport by immune signaling in Arabidopsis.

Soil availability of inorganic ortho-phosphate (PO43-, Pi) is a key determinant of plant growth and fitness.1 Plants regulate the capacity of their roots to take up inorganic phosphate by adapting the abundance of H+-coupled phosphate transporters of the PHOSPHATE TRANSPORTER 1 (PHT1) family2 at the plasma membrane (PM) through transcriptional and post-translational changes driven by the genetic network of the phosphate starvation response (PSR).3-8 Increasing evidence also shows that plants integrate immune responses to alleviate phosphate starvation stress through the association with beneficial microbes.9-11 Whether and how such phosphate transport is regulated upon activation of immune responses is yet uncharacterized. To address this question, we first developed quantitative assays based on changes in the electrical PM potential to measure active Pi transport in roots in real time. By inserting micro-electrodes into bulging root hairs, we were able to determine key characteristics of phosphate transport in intact Arabidopsis thaliana (hereafter Arabidopsis) seedlings. The fast Pi-induced depolarization observed was dependent on the activity of the major phosphate transporter PHT1;4. Notably, we observed that this PHT1;4-mediated phosphate uptake is repressed upon activation of pattern-triggered immunity. This inhibition depended on the receptor-like cytoplasmic kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBS1-LIKE KINASE 1 (PBL1), which both phosphorylated PHT1;4. As a corollary to this negative regulation of phosphate transport by immune signaling, we found that PHT1;4-mediated phosphate uptake normally negatively regulates anti-bacterial immunity in roots. Collectively, our results reveal a mechanism linking plant immunity and phosphate homeostasis, with BIK1/PBL1 providing a molecular integration point between these two important pathways.

The GABA transaminase GabT is required for full virulence of Ralstonia solanacearum in tomato.

The utilization of plant gamma-aminobutyric acid (GABA) is essential for the pathogenicity of the bacterial plant pathogen Ralstonia solanacearum. A knockout mutant in the GABA transaminase-encoding gene gabT is unable to utilize GABA as a nutrient and its ability to cause disease in plants is strongly compromised. However, the proximity of the gabD gene (encoding a succinate semialdehyde dehydrogenase) in the same operon raises the question of whether an impact on the gabD gene underlies or contributes to the virulence attenuation of the ΔgabT mutant. In this work, we use genetic complementation to show that the expression of the gabT gene is able to rescue the impaired virulence of the ΔgabT knockout mutant in tomato plants, confirming that the gabT-encoded GABA transaminase is indeed required for full virulence of R. solanacearum in a natural host plant.

Tomato Stem Injection for the Precise Assessment of Ralstonia solanacearum Fitness in Planta.

Ralstonia solanacearum is a soil-borne pathogen with worldwide distribution that causes bacterial wilt disease in more than 250 plant species. R. solanacearum invades plants through the roots, reaches the vascular system, and colonizes the whole plant by moving through the xylem, where it eventually replicates rapidly, causing plant death. Usual assays to measure the virulence of R. solanacearum under laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a protocol to assess the replication of R. solanacearum following injection into tomato stems. This protocol includes four major steps: 1) growth of tomato plants; 2) R. solanacearum injection into tomato stems; 3) collection of tomato xylem samples and bacterial quantitation; and 4) data analysis and representation. This method bypasses the natural penetration process of the pathogen, thus minimizing variation associated with stochastic events during bacterial invasion, and provides a sensitive and accurate measurement of bacterial fitness inside xylem vessels.

Activation loop phosphorylation of a non-RD receptor kinase initiates plant innate immune signaling.

Receptor kinases (RKs) are fundamental for extracellular sensing and regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) are primarily peptide receptors that regulate responses to myriad internal and external stimuli. Phosphorylation of LRR-RK cytoplasmic domains is among the earliest responses following ligand perception, and reciprocal transphosphorylation between a receptor and its coreceptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in antibacterial immunity. These results reveal a noncatalytic role for EFR in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RKs with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor, which could initiate signaling either allosterically or through driving the dissociation of negative regulators of the complex.

A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves.

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to analyze the replication of R. solanacearum upon infiltration into the leaves of Nicotiana benthamiana, in which gene expression has been altered using Agrobacterium tumefaciens. The protocol includes five major steps: 1) growth of N. benthamiana plants; 2) infiltration of A. tumefaciens; 3) R. solanacearum inoculation; 4) sample collection and bacterial quantitation; 5) data analysis and representation. The transient gene expression or gene silencing prior to R. solanacearum inoculation provides a straightforward way to perform genetic analysis of plant functions involved in the interaction between pathogen and host, using the appropriate combination of A. tumefaciens and R. solanacearum strains, with high sensitivity and accuracy provided by the quantitation of bacterial numbers in plant tissues.