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.

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.

Intra-strain Elicitation and Suppression of Plant Immunity by Ralstonia solanacearum Type-III Effectors in Nicotiana benthamiana.

Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. We further found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum secretes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by plant immune system.

A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity.

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.

Preparation of Fe3O4@C@PANI magnetic microspheres for the extraction and analysis of phenolic compounds in water samples by gas chromatography-mass spectrometry.

In this work, core-shell structure Fe(3)O(4)@C@polyaniline magnetic microspheres were synthesized using simple hydrothermal reactions. The carbon-coated magnetic microspheres (Fe(3)O(4)@C) were first synthesized by a hydrothermal reaction, and then aniline was polymerized on the magnetic core via another hydrothermal reaction. Then, the obtained Fe(3)O(4)@C@polyaniline magnetic microspheres were applied as magnetic adsorbents for the extraction of aromatic molecules due to π-π interactions between polyaniline shell and aromatic compounds. In our study, five kinds of phenols including phenol, 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP), pentachlorophenol (PCP) and bisphenol A (BPA) were selected as the model analytes to verify the extraction ability of Fe(3)O(4)@C@PANI microspheres. After derivatization, the phenols were detected using gas chromatography-mass spectrometry (GC-MS). The dominant parameters affecting enrichment efficiency were investigated and optimized. Under the optimal conditions, the proposed method was evaluated, and applied to the analysis of phenols in real water samples. The results demonstrated that our proposed method based on Fe(3)O(4)@C@polyaniline magnetic microspheres had good linearity (r(2)>0.991), and limits of quantification (2.52-29.7 ng/mL), high repeatability (RSD<13.1%) and good recovery (85.3-110.6%).