Yves Marco

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.

The Arabidopsis Cytosolic Thioredoxin h5 Gene Induction by Oxidative Stress and Its W-Box-Mediated Response to Pathogen Elicitor

The AtTRXh5 protein belongs to the cytosolic thioredoxins h family that, in Arabidopsis, contains eight members showing very distinct patterns and levels of expression. Here, we show that the AtTRXh5 gene is up-regulated during wounding, abscission, and senescence, as well as during incompatible interactions with the bacterial pathogen Pseudomonas syringae. By electrophoretic mobility shift assays, a binding activity on a W-box in the AtTRXh5 promoter region was found induced by treatments with the P. syringae-derived elicitor peptide flg22, suggesting that a WRKY transcription factor controls AtTRXh5 induction upon elicitor treatment. Remarkably, AtTRXh5 was up-regulated in plants overexpressing WRKY6. More generally, AtTRXh5 is induced in response to oxidative stress conditions. Collectively, our data indicate a possible implication of the cytosolic thioredoxin AtTRXh5 in response to pathogens and to oxidative stresses. In addition, this regulation is unique to AtTRXh5 among the thioredoxin h family, arguing in favor of a speciation rather than to a redundancy of the members of this multigenic family.

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.

Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria

During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified. In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented. Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity). Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation. The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P. solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development. Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.

The ERECTA Receptor-Like Kinase Regulates Cell Wall–Mediated Resistance to Pathogens in Arabidopsis thaliana

Some receptor-like kinases (RLK) control plant development while others regulate immunity. The Arabidopsis ERECTA (ER) RLK regulates both biological processes. To discover specific components of ER-mediated immunity, a genetic screen was conducted to identify suppressors of erecta (ser) susceptibility to Plectosphaerella cucumerina fungus. The ser1 and ser2 mutations restored disease resistance to this pathogen to wild-type levels in the er-1 background but failed to suppress er-associated developmental phenotypes. The deposition of callose upon P. cucumerina inoculation, which was impaired in the er-1 plants, was also restored to near wild-type levels in the ser er-1 mutants. Analyses of er cell walls revealed that total neutral sugars were reduced and uronic acids increased relative to those of wild-type walls. Interestingly, in the ser er-1 walls, neutral sugars were elevated and uronic acids were reduced relative to both er-1 and wild-type plants. The cell-wall changes found in er-1 and the ser er-1 mutants are unlikely to contribute to their developmental alterations. However, they may influence disease resistance, as a positive correlation was found between uronic acids content and resistance to P. cucumerina. We propose a specific function for ER in regulating cell wall-mediated disease resistance that is distinct from its role in development.

Delayed symptom development in ein2-1, an Arabidopsis ethylene-insensitive mutant, in response to bacterial wilt caused by Ralstonia solanacearum

ABSTRACT Wilt disease caused by the phytopathogenic bacterium Ralstonia solanacearum is poorly understood at the molecular level. The possible roles of salicylic acid, jasmonic acid, and ethylene, compounds commonly associated with the plant response to pathogens, in wilt symptom development were investigated using various Arabidopsis thaliana mutants in a Col-0 background, an ecotype that develops wilt symptoms in response to the virulent GMI1000 strain. Following root inoculation, wilt symptoms were delayed in ein2-1, an ethylene-insensitive mutant, in response to several virulent strains of the pathogen. In ein2-1, bacteria invade the plant and multiply, reaching concentrations slightly lower than those detected in susceptible plants but 1 to 2 logs higher than in Nd-1, an A. thaliana ecotype resistant to strain GMI1000. This delay in disease symptom development of ein2-1 plants suggests that ethylene signaling plays a critical role in wilt disease development. Furthermore, a strong accumulation of transcripts corresponding to PR-3 and PR-4, two ethylene-responsive genes, was observed in susceptible Col-0 plants, but not in ein2-1 and Nd-1 plants, providing additional evidence for a role of ethylene in wilt symptom production. However, this hormone is probably not involved in the establishment of resistance to R. solanacearum, because homozygous ein2-1 plants in a resistant background remain fully resistant to strain GMI1000.