Structural identification of the O-antigen fraction from the lipopolysaccharide of the Burkholderia ambifaria strain 19182.

The Burkholderia cepacia complex comprises a group of bacterial strains with both beneficial and detrimental effects to plant and animals. Gram negative bacterial lipopolysaccharide is one of the most important molecular factors involved in the dialogue between the microbe and the host and in this context we have isolated and identified the O-antigen fraction of the Burkholderia ambifaria strain 19182. It consists of two different O-polysaccharides built up on 6-deoxy sugars, among which the 6-deoxy-altrose in the d absolute configuration, is present. This monosaccharide is found for the first time and it is a unique feature associated to this strain.

Discrimination of Arabidopsis PAD4 activities in defense against green peach aphid and pathogens.

The Arabidopsis (Arabidopsis thaliana) lipase-like protein PHYTOALEXIN DEFICIENT4 (PAD4) is essential for defense against green peach aphid (GPA; Myzus persicae) and the pathogens Pseudomonas syringae and Hyaloperonospora arabidopsidis. In basal resistance to virulent strains of P. syringae and H. arabidopsidis, PAD4 functions together with its interacting partner ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defenses. By contrast, dissociated forms of PAD4 and EDS1 signal effector-triggered immunity to avirulent strains of these pathogens. PAD4-controlled defense against GPA requires neither EDS1 nor SA. Here, we show that resistance to GPA is unaltered in an eds1 salicylic acid induction deficient2 (sid2) double mutant, indicating that redundancy between EDS1 and SID2-dependent SA, previously reported for effector-triggered immunity conditioned by certain nucleotide-binding-leucine-rich repeat receptors, does not explain the dispensability of EDS1 and SID2 in defense against GPA. Mutation of a conserved serine (S118) in the predicted lipase catalytic triad of PAD4 abolished PAD4-conditioned antibiosis and deterrence against GPA feeding, but S118 was dispensable for deterring GPA settling and promoting senescence in GPA-infested plants as well as for pathogen resistance. These results highlight distinct molecular activities of PAD4 determining particular aspects of defense against aphids and pathogens.

Different roles of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity.

• Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. • The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. • EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. • Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.

Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves.

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.

The RAP1 gene confers effective, race-specific resistance to the pea aphid in Medicago truncatula independent of the hypersensitive reaction.

Plant resistance to pathogens is commonly associated with a hypersensitive response (HR), but the degree to which the HR is responsible for incompatibility is subject to debate. Resistance to aphids is likely to share features with resistance to pathogens but is less well understood. Here, we report effective resistance to the pea aphid Acyrthosiphon pisum in Medicago truncatula. Aphids lost weight and died rapidly (within two days) on the resistant genotype Jemalong, which developed necrotic lesions following infestation. Lesions were induced by nonvascular intracellular stylet punctures by aphids, remained localized to the site of stylet entry, stained for the presence of reactive oxygen species, and were similar to the HR induced by the bacterial pathogen Pseudomonas syringae pv. phaseolicola. The implication that aphid-induced lesions confer resistance was tested by quantitative trait loci analysis using recombinant inbred lines derived from a cross between Jemalong and the susceptible genotype DZA315.16. One major locus, RAP1, was identified that was sufficient to confer race-specific resistance against the pea aphid and was mapped to the middle of chromosome 3. Surprisingly, a separate locus, mapping to the top of chromosome 3, governed aphid-induced HR, indicating that the HR-like lesions are not required for RAP1-mediated aphid resistance.

Enterohemorrhagic Escherichia coli exploits EspA filaments for attachment to salad leaves.

Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.

Phloem-based resistance to green peach aphid is controlled by Arabidopsis PHYTOALEXIN DEFICIENT4 without its signaling partner ENHANCED DISEASE SUSCEPTIBILITY1.

Green peach aphid (GPA) Myzus persicae (Sülzer) is a phloem-feeding insect with an exceptionally wide host range. Previously, it has been shown that Arabidopsis thaliana PHYTOALEXIN DEFICIENT4 (PAD4), which is expressed at elevated levels in response to GPA infestation, is required for resistance to GPA in the Arabidopsis accession Columbia. We demonstrate here that the role of PAD4 in the response to GPA is conserved in Arabidopsis accessions Wassilewskija and Landsberg erecta. Electrical monitoring of aphid feeding behavior revealed that PAD4 modulates a phloem-based defense mechanism against GPA. GPA spends more time actively feeding from the sieve elements of pad4 mutants than from wild-type plants, and less time feeding on transgenic plants in which PAD4 is ectopically expressed. The activity of PAD4 in limiting phloem sap uptake serves as a deterrent in host-plant choice, and restricts aphid population size. In Arabidopsis defense against pathogens, all known PAD4 functions require its signaling and stabilizing partner EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1). Bioassays with eds1 mutants alone or in combination with pad4 and with plants conditionally expressing PAD4 under the control of a dexamethasone-inducible promoter reveal that PAD4-modulated defense against GPA does not involve EDS1. Thus, a PAD4 mode of action that is uncoupled from EDS1 determines the extent of aphid feeding in the phloem.

Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity.

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.

Plant immunity: the EDS1 regulatory node.

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its interacting partner, PHYTOALEXIN DEFICIENT 4 (PAD4), constitute a regulatory hub that is essential for basal resistance to invasive biotrophic and hemi-biotrophic pathogens. EDS1 and PAD4 are also recruited by Toll-Interleukin-1 receptor (TIR)-type nucleotide binding-leucine rich repeat (NB-LRR) proteins to signal isolate-specific pathogen recognition. Recent work points to a fundamental role of EDS1 and PAD4 in transducing redox signals in response to certain biotic and abiotic stresses. These intracellular proteins are important activators of salicylic acid (SA) signaling and also mediate antagonism between the jasmonic acid (JA) and ethylene (ET) defense response pathways. EDS1 forms several molecularly and spatially distinct complexes with PAD4 and a newly discovered in vivo signaling partner, SENESCENCE ASSOCIATED GENE 101 (SAG101). Together, EDS1, PAD4 and SAG101 provide a major barrier to infection by both host-adapted and non-host pathogens.

An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus.

In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide-binding site, leucine-rich-repeats and amino-terminal similarity to animal Toll and Interleukin-1 (so-called TIR-NBS-LRR proteins). EDS1 is not required by NBS-LRR proteins that possess an amino-terminal coiled-coil motif (CC-NBS-LRR proteins). Using virus-induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR-NBS-LRR protein, was EDS1-dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC-NBS-LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR-NBS-LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.