Laurent Deslandes

Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus

RRS1-R confers broad-spectrum resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum. Although genetically defined as recessive, this R gene encodes a protein whose structure combines the TIR-NBS-LRR domains found in several R proteins and a WRKY motif characteristic of some plant transcriptional factors and behaves as a dominant gene in transgenic susceptible plants. Here we show that PopP2, a R. solanacearum type III effector, which belongs to the YopJ/AvrRxv protein family, is the avirulence protein recognized by RRS1-R. Furthermore, an interaction between PopP2 and both RRS1-R and RRS1-S, present in the resistant Nd-1 and susceptible Col-5 Arabidopsis thaliana ecotypes, respectively, was detected by using the yeast split-ubiquitin two-hybrid system. This interaction, which required the full-length R protein, was not observed between the RRS1 proteins and PopP1, another member of the YopJ/AvrRxv family present in strain GMI1000 and that confers avirulence in Petunia. We further demonstrate that both the Avr protein and the RRS1 proteins colocalize in the nucleus and that the nuclear localization of the RRS1 proteins are dependent on the presence of PopP2.

Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes.

The identification of two Arabidopsis thaliana genes involved in determining recessive resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum, is reported. Dominant (RRS1-S) and recessive (RRS1-R) alleles from susceptible and resistant accessions encode highly similar predicted proteins differing in length and which present a novel structure combining domains found in plant Toll-IL-1 receptor–nucleotide binding site–leucin-rich repeat resistance proteins and a WRKY motif characteristic of some plant transcriptional factors. Although genetically defined as a recessive allele, RRS1-R behaves as a dominant resistance gene in transgenic plants. Sequence analysis of the RRS1 genes present in two homozygous intragenic recombinant lines indicates that several domains of RRS1-R are essential for its resistance function. Additionally, RRS1-R-mediated resistance is partially salicylic acid- and NDR1-dependent, suggesting the existence of similar signaling pathways to those controlled by resistance genes in specific resistance.

Impairment of Cellulose Synthases Required for Arabidopsis Secondary Cell Wall Formation Enhances Disease Resistance

Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane–localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW–RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)–responsive, defense-related genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens.

Arabidopsis EDS1 Connects Pathogen Effector Recognition to Cell Compartment–Specific Immune Responses

Coordination of different defense pathways across cell compartments produces a fully effective innate immune response. Pathogen effectors are intercepted by plant intracellular nucleotide binding–leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll–interleukin-1 receptor)–NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment–specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

A remorin protein interacts with symbiotic receptors and regulates bacterial infection

Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.

Catch me if you can: bacterial effectors and plant targets

To suppress plant defense responses and favor the establishment of disease, phytopathogenic bacteria have gained the ability to deliver effector molecules inside host cells through the type III secretion system. Inside plant cells, bacterial effector proteins may be addressed to different subcellular compartments where they are able to manipulate a variety of host cellular components and molecular functions. Here we review how the recent identification and functional characterization of plant components targeted by bacterial effectors, as well as the discovery of new pathogen recognition capabilities evolved in turn by plant cells, have significantly contributed to further our knowledge about the intricate molecular interactions that are established between plants and their invading bacteria.

A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity.

Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

RD19, an Arabidopsis Cysteine Protease Required for RRS1-R–Mediated Resistance, Is Relocalized to the Nucleus by the Ralstonia solanacearum PopP2 Effector

Bacterial wilt, a disease impacting cultivated crops worldwide, is caused by the pathogenic bacterium Ralstonia solanacearum. PopP2 (for Pseudomonas outer protein P2) is an R. solanacearum type III effector that belongs to the YopJ/AvrRxv protein family and interacts with the Arabidopsis thaliana RESISTANT TO RALSTONIA SOLANACEARUM 1-R (RRS1-R) resistance protein. RRS1-R contains the Toll/Interleukin1 receptor–nucleotide binding site–Leu-rich repeat domains found in several cytoplasmic R proteins and a C-terminal WRKY DNA binding domain. In this study, we identified the Arabidopsis Cys protease RESPONSIVE TO DEHYDRATION19 (RD19) as being a PopP2-interacting protein whose expression is induced during infection by R. solanacearum. An Arabidopsis rd19 mutant in an RRS1-R genetic background is compromised in resistance to the bacterium, indicating that RD19 is required for RRS1-R–mediated resistance. RD19 normally localizes in mobile vacuole-associated compartments and, upon coexpression with PopP2, is specifically relocalized to the plant nucleus, where the two proteins physically interact. No direct physical interaction between RRS1-R and RD19 in the presence of PopP2 was detected in the nucleus as determined by Förster resonance energy transfer. We propose that RD19 associates with PopP2 to form a nuclear complex that is required for activation of the RRS1-R–mediated resistance response.

Genetic characterization of RRS1, a recessive locus in Arabidopsis thaliana that confers resistance to the bacterial soilborne pathogen Ralstonia solanacearum.

The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systematically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.

Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as “guards”. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.