Plants and animals mediate early steps of the innate immune response through pathogen recognition receptors (PRRs). PRRs commonly associate with or contain members of a monophyletic group of kinases called the interleukin-1 receptor-associated kinase (IRAK) family that include Drosophila Pelle, human IRAKs, rice XA21 and Arabidopsis FLS2. In mammals, PRRs can also associate with members of the receptor-interacting protein (RIP) kinase family, distant relatives to the IRAK family. Some IRAK and RIP family kinases fall into a small functional class of kinases termed non-RD, many of which do not autophosphorylate the activation loop. We surveyed the yeast, fly, worm, human, Arabidopsis, and rice kinomes (3,723 kinases) and found that despite the small number of non-RD kinases in these genomes (9%-29%), 12 of 15 kinases known or predicted to function in PRR signaling fall into the non-RD class. These data indicate that kinases associated with PRRs can largely be predicted by the lack of a single conserved residue and reveal new potential plant PRR subfamilies.

ABSTRACT The ability of some phytopathogenic bacterial strains to inhibit the growth of others in mixed infections has been well documented. Here we report that such antagonistic interactions occur between several wild-type strains of the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. In mixed inoculations, a wild-type Philippine strain was found to inhibit the growth of a wild-type Korean strain. Furthermore, a nonpathogenic mutant of the Philippine strain maintained these antagonistic properties. Growth curve analysis indicated that both the wild-type Philippine strain and its nonpathogenic mutant inhibited the growth of the Korean strain 2 days after infection and prior to the onset of disease symptoms. When mixed with the nonpathogenic mutant, 10 out of 18 diverse wild-type X. oryzae pv. oryzae strains did not cause disease. Conversely, three of the strains that were not affected by the nonpathogenic mutant were found to inhibit the growth of both the wild-type and mutant Philippine strains, indicating that antagonism is widespread and strain specific. The observed growth inhibition occurred only in planta and did not correlate with bacteriocin activity in vitro. Antagonistic interactions also were found to affect resistance (R) gene-mediated resistance. The R gene Xa21 was capable of protecting rice plants coinoculated with nonantagonistic virulent and avirulent strains; however, when avirulent strains were coinoculated with virulent antagonistic strains, disease ensued. Taken together, these results indicate that X. oryzae pv. oryzae has evolved strategies to compete with rival strains in a fashion that allows virulent strains to evade R gene-mediated protection even when avirulent strains are present in the inoculum.

The rice (Oryza sativa) genome contains 1,429 protein kinases, the vast majority of which have unknown functions. We created a phylogenomic database (http://rkd.ucdavis.edu) to facilitate functional analysis of this large gene family. Sequence and genomic data, including gene expression data and protein-protein interaction maps, can be displayed for each selected kinase in the context of a phylogenetic tree allowing for comparative analysis both within and between large kinase subfamilies. Interaction maps are easily accessed through links and displayed using Cytoscape, an open source software platform. Chromosomal distribution of all rice kinases can also be explored via an interactive interface.

Determination of gene function is particularly problematic when studying large-gene families because redundancy limits the ability to assess the contributions of individual genes experimentally. Phylogenomics is a phylogenetic approach used in comparative genomics to predict the biological functions of members of large gene-families by assessing the similarity among gene products. In this report, we describe the application of the Rice Kinase Database for elucidating functions of individual members of this gene family.

Determination of gene function is particularly problematic when studying large-gene families because redundancy limits the ability to assess the contributions of individual genes experimentally. Phylogenomics is a phylogenetic approach used in comparative genomics to predict the biological functions of members of large gene-families by assessing the similarity among gene products. In this report, we describe the application of the Rice Kinase Database for elucidating functions of individual members of this gene family.

The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. Here, we report that XA21 binds to a WRKY transcription factor, called OsWRKY62. The OsWRKY62 gene encodes two splice variants (OsWRKY62.1 and OsWRKY62.2). OsWRKY62.1:smGFP2 and OsWRKY62.2:smGFP2 fusion proteins partially localize to the nucleus. Transgenic plants overexpressing OsWRKY62.1 are compromised in basal defense and Xa21-mediated resistance to Xoo. Furthermore, overexpression of OsWRKY62.1 suppresses the activation of defense-related genes. These results imply that OsWRKY62 functions as a negative regulator of innate immunity in rice, and serves as a critical mediator of both basal and race-specific defense responses.

Innate immunity to microorganisms relies on the specific sensing of pathogen-associated molecules by host recognition receptors. Whereas studies in animals have largely focused on the recognition of extracellular pathogen-associated molecules by the TLR (toll-like receptor) superfamily, few studies have been carried out in plants, and it is not understood how these molecules are secreted or modified. The rice Xa21 gene encodes a receptor-like kinase that provides immunity against strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae carrying AvrXa21 activity. We identified four X. oryzae pv. oryzae genes that are required for AvrXa21 activity. raxA, raxB, and raxC encode proteins with similarity to a membrane fusion protein, an ATP-binding cassette transporter, and an outer membrane protein, respectively, of bacterial type I secretion systems. The fourth gene, raxST, encodes a sulfotransferase-like protein. Sequence analysis of three naturally occurring X. oryzae pv. oryzae strains no longer recognized by Xa21 revealed alterations in the raxST and raxA genes. The raxC gene complemented an Escherichia coli tolC mutant for secretion of a double glycine-leader peptide confirming the function of raxC in type I secretion. These results indicate that bacterial type I secretion is necessary for Xa21-mediated recognition and immunity and further suggest that type I secretion and modification of pathogen-associated molecules play an important role in triggering the innate immune response in rice.

Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Kinases that are associated with the receptors or are part of the receptors themselves modulate signaling through phosphorylation events. The rice (Oryza sativa L.) XA21 receptor kinase is a key recognition and signaling determinant in the innate immune response. A yeast two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as bait, identified a protein phosphatase 2C (PP2C), called XA21 binding protein 15 (XB15). The interaction of XA21 and XB15 was confirmed in vitro and in vivo by glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, respectively. XB15 fusion proteins purified from Escherichia coli and from transgenic rice carry PP2C activity. Autophosphorylated XA21 can be dephosphorylated by XB15 in a temporal- and dosage-dependent manner. A serine residue in the XA21 JM domain is required for XB15 binding. Xb15 mutants display a severe cell death phenotype, induction of pathogenesis-related genes, and enhanced XA21-mediated resistance. Overexpression of Xb15 in an XA21 rice line compromises resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. These results demonstrate that Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response.

Background

Results

Conclusion