The rice receptor-like kinase, XA21, is a key recognition determinant in the innate immune response, a pathogen defense pathway widely conserved between plants and animals. Few proteins that transduce the rice innate immune response have been characterized. For this reason the isolation of a protein from an in vivo XA21 complex, called BiP (luminal binding protein, also called glucose regulated protein 78 (GRP78)), is particularly important. BiP is an endoplasmic reticulum (ER)-located member of the family of heat shock protein (HSP) 70 chaperones. In BiP3-overexpressing rice plants, XA21-mediated resistance is compromised and XA21 stability is significantly decreased. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance.

The long-term goal of this project is to elucidate the mechanisms of innate immunity, determine the role of BiP in these signaling pathways, and use this information to develop new strategies for disease control in plants and animals. The project will use biochemical, cell biological and genetic approaches to determine how the ER chaperone BiP affects XA21-mediated signaling. Results from these studies will lead to major advances in understanding biogenesis and processing of plant and animal sensors of pathogen-derived molecules. Because XA21 is closely related to sensors controlling innate immunity in other plants and animals, the expected results will have a broad impact on understanding and controlling diseases of cereals and humans. The results will have relevance to other plant-microbe interactions and will be disseminated broadly to enhance scientific and technological understanding of the mechanisms of innate immunity.

Postdoctoral and graduate students will receive training in bacterial genetics, biochemistry, and plant pathology. Educational outreach includes a month-long science and art program to be presented in a local elementary school.

Project Report

Plants recognize conserved microbial signatures via pattern recognition receptors (PRRs). Well-characterized PRRs include the Arabidopsis flagellin sensitive 2 (FLS2) receptor, the Arabidopsis elongation factor Tu receptor (EFR), and the rice XA21 (Xanthomonas resistance 21) receptor. These proteins share a similar structure: an extracellular leucine-rich repeat (LRR) domain, a transmembrane (TM) domain, and an intracellular non-arginine-aspartate (non-RD) kinase domain. The non-RD kinase motif is a hallmark of kinases involved in the initial stages of PRR-mediated immunity. In contrast, RD kinases are associated with a function in developmental processes. Rice XA3 (also known as XA26) also shares this LRR-TM-non-RD kinase domain structure. XA3 also confers another important trait characteristic of PRRs: broad-spectrum resistance. For these reasons, XA3 is predicted to be a PRR. Membrane-bound PRRs are synthesized in the endoplasmic reticulum (ER) where they are subject to ER-quality control. The ER-quality control is a conserved process in eukaryotic cells responsible for monitoring correct folding and processing of membrane and secretory proteins. Many ER proteins, including HSP70 luminal-binding protein (BiP), HSP40 ERdj3B, stromal-derived factor 2 (SDF2), calreticulin3 (CRT3), UDP-glucose glycoprotein glucosyl transferase (UGGT), and ER retention defective 2B (ERD2B), are involved in ER-quality control. Recent research suggests that the ER-resident chaperone BiP, an integral protein of the ER quality control system, is one of the main chaperones regulating biogenesis and degradation of membrane-resident PRRs. For example, in BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo. In Arabidopsis, a large ER chaperone complex BiP/ ERdj3B/SDF2 is required for the proper accumulation of EFR, further supporting a role for ER-quality control in membrane-resident PRR-mediated function. A differential requirement for genes governing ER-quality control has been observed in structurally-related receptors. For example, Arabidopsis FLS2-mediated responses are not impaired in the mutants, sdf2, crt3, uggt, and erd2b. In contrast, the genes which encode ER proteins, are all required for EFR-mediated signal transduction. Similarly, overexpression of BiP3 in rice does not affect rice brassinosteroid-insensitive 1 (OsBRI1)-mediated signaling, even though OsBRI1 shows an overall structural similarity with XA21. In contrast to XA21, OsBRI1 carries the RD class of kinases and regulates growth and developmental responses. As observed for rice, Arabidopsis BiPs fail to interact with wild-type BRI1. In animals, processing of Toll-like receptors (TLRs), which are key determinants of the innate immune response, also require specific ER chaperones. For example, despite of its role as a general housekeeping chaperone, mouse ER gp96 is specific for processing TLR2, 4, 5, 7, and 9 in macrophages. Although gp96-deficient macrophages fail to respond to flagellin, the ligand for TLR5, mutant macrophages display normal development and activation by interferon-c, tumor necrosis factor-a, and interleukin-1b.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Michael L. Mishkind
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University of California Davis
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