Ofir Bahar

The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

A sulfated peptide activates a rice immune receptor. Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.

Tyrosine sulfation in a Gram-negative bacterium

Tyrosine sulfation, a well-characterized post-translation modification in eukaryotes, has not previously been reported in prokaryotes. Here, we demonstrate that the RaxST protein from the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae, is a tyrosine sulfotransferase. We used a newly developed sulfotransferase assay and ultraviolet photodissociation mass spectrometry to demonstrate that RaxST catalyses sulfation of tyrosine 22 of the Xoo Ax21 (activator of XA21-mediated immunity) protein. These results demonstrate a previously undescribed post-translational modification in a prokaryotic species with implications for studies of host immune responses and bacterial cell-cell communication systems.

The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

Pattern recognition receptors (PRRs) play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec) system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

Small Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium

The rice XA21 pattern recognition receptor binds a type I secreted sulfated peptide, called axYS22, derived from the Ax21 (activator of XA21-mediated immunity) protein. The conservation of Ax21 in all sequenced Xanthomonas spp. and closely related genera suggests that Ax21 serves a key biological function. Here we show that the predicted N-terminal sequence of Ax21 is cleaved prior to secretion outside the cell and that mature Ax21 serves as a quorum sensing (QS) factor in Xanthomonas oryzae pv. oryzae. Ax21-mediated QS controls motility, biofilm formation and virulence. We provide genetic evidence that the Xoo RaxH histidine kinase serves as the bacterial receptor for Ax21. This work establishes a critical role for small protein-mediated QS in a Gram-negative bacterium.

Bacterial Outer Membrane Vesicles Induce Plant Immune Responses

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

Retraction: Small Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium

The authors retract this publication due to concerns about the integrity of strain PXO99Dax21 employed in the study. During additional experiments carried out at our laboratory, we discovered that the strain PXO99Dax21 employed in the study was mixed up with another strain in our collection. This compromises the validity of Figures 1, 2 and 3 in the article as we are unsure as to whether the correct strain was employed in the studies. We have taken steps to repeat the experiments but we have been unable to validate the knockout mutants generated so far. We have also attempted to replicate the work using strains obtained from another group, but in our hands, those strains did not infect rice plants.

Bacterial outer membrane vesicles at the plant–pathogen interface

Gram-negative bacteria outer membrane vesicles (OMVs) are extracellularly released blebs, constantly detaching from the bacterial cell surface. Being ubiquitous among bacteria and diverse in content, OMVs have a plethora of functions: promoting virulence, mediating bacterial cell–cell communication, modulating host immune response, and more. Though most research on OMVs has been carried out on animal pathogens, production of OMVs by plant pathogenic bacteria is predicted to be similarly intrinsic to their biology. Recent studies in the field of plant–bacteria interactions have begun to unravel the roles of OMVs, showing their involvement in biofilm formation, virulence, and modulation of plant immunity. With a range of general to highly specialized roles, these structures can act as an adaptive toolbox during pathogenesis and stress. This Pearl will crystallize current OMV research with a special focus on the role OMVs play in plant–bacteria interactions.