Wen-Yuan Song - US grants

The long-term goal of this project is to gain an understanding of receptor serine/threonine kinase-mediated signaling in plants. Receptor kinases, including receptor tyrosine kinases and receptor serine/threonine kinases, are major transmembrane signal transducers of many processes such as cell proliferation, differentiation, migration, apoptosis, and defense. In animal systems, receptor tyrosine kinases appear to be predominant in cell signaling. In plants, almost all receptor-like kinases (RLK) identified have serine/threonine specificity, making plants particularly amenable to studying receptor serine/threonine kinase-mediated signaling. The first rice disease resistance gene Xa21, which encodes a RLK, was cloned. The putative intracellular domain of XA21 can autophosphorylate multiple serine and threonine residues and interacts with seven proteins (XBs) in a yeast two-hybrid system. One of the seven binding proteins, XB1, physically interacts with and acts as a substrate of the XA21 kinase. XB1 consists of two domains: an ankyrin domain involved in interacting with XA21 and a RING finger motif belonging to the newly defined R-box family that may play a universal role in ubiquitin-mediated protein degradation. These studies provide an excellent system to study mechanisms underlying initiation, propagation, and termination of RLK-mediated signaling. The specific goals of this project are:

1. Investigate the mode of regulation of the XA21-mediated signaling
2. Investigate the role of XBs in the XA21-mediated signaling

Achieving these goals will provide insights into RLK-mediated signaling and will advance our understanding of plant disease resistance at the molecular level. Information from these studies would provide new approaches for the engineering of broad-spectrum, durable disease resistance in rice and other crop plants, thereby reducing the dependency of crop production on environmentally unfriendly chemicals.

The cereals, including rice, maize, wheat, barley, sorghum, and the millets, are the most important group of plants in agriculture, and environmental stress is the major limitation to their productivity. Increasing their tolerance to abiotic and biotic stress is an important goal in agriculture and considerable progress has been made in discovering the mechanisms of cell signaling that regulate the response to these stresses in plants. This project will dramatically enhance this progress by generating a protein-protein interaction database for 275 rice protein kinases, that represent each of the protein kinase subfamilies present in the recently sequenced rice genome. The protein interaction data will be obtained using both the yeast two hybrid method and a method utilizing mass spectrometry analysis of affinity-tagged protein complexes that will be purified from transgenic plants. Additionally, 100 representative orotein kinases will be mutated by either insertional mutations or RNAi silencing, and their mutant phenotypes analyzed. This combined information will available at the PlantsP website and will advance the understanding of cell signaling pathways. The knowledge of the mechanisms that plant cells use to regulate their tolerance to environmental stress will be critical for improving the agricultural productivity of the cereals.
Deliveables:
Data.
The project data will be deposited at the PlantsP website (http://plantsp.sdsc.edu) along with the gene annotations. Full-length cDNA sequences will be deposited in Genbank and PlantsP as soon as they are completed and checked. The protein interaction data, and knockout phenotype data will be entered into the PlantsP database at no longer than six month intervals after completing the individual experiments.

Materials.
The rice cDNA libraries, BD-kinase constructs, and yeast 2-hybrid bait arrays will be made available on request from the Song laboratory (Wen-Yuan Song, University of Florida). They will be advertised in a publication, which will occur in year. Availability of the libraries will also be advertised on the PlantsP website and at the annual plant phosphorylation meeting, which reaches most of the plant protein kinase community.

Transgenic rice seeds will be made available from Pam Ronald's laboratory (UC Davis) or Jian-Kang Zhu's laboratory (Univeristy of Arizona) until a suitable rice resource center is available.

Defense against bacterial disease is a costly undertaking by plants. Overamplification or dysregulation of the immune system often causes deleterious effects. Therefore, tight control of immune sensors and restriction of the immune response to pathogen-infected tissues are thought to be crucial for survival of the host plant. The rice cell-surface immune sensor XA21confers resistance to water-deficit stress and immunity against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). This project will study the regulation of XA21 by a protein capable of cleaving XA21 in the cell membrane. The knowledge gained will not only contribute to agricultural applications but will also create training opportunities for a postdoctoral scholar, graduate and undergraduate students, precollege students and a total of 24 educators from Title I high schools (containing minority and socioeconomically challenged student populations of 40% or greater). By engaging in this project, the participants will learn cutting-edge scientific ideas and methods and contribute to the discovery of knowledge that may have broad implications. The participating teachers will gain interest in and understanding of general and advanced molecular techniques. As a result, this knowledge and interest will ultimately be shared with high school students in their classrooms during the span of this project. The exposure of younger researchers and students to the crop rice and its diseases will help citizens better understand agriculture and food security.

Intramembrane proteolysis is an evolutionarily conserved cellular mechanism that mediates cleavage of transmembrane proteins within the lipid bilayers. However, very little is known about this important regulatory process in plants, largely due to lack of known biological substrates of the widespread intramembrane proteases. The intramembrane protease Limiting XA21 to Leaf 1 (LXL1) is capable of cleaving XA21 in planta. This project aims to verify XA21 as a biological substrate of LXL1 in rice, to pinpoint the LXL1 cleavage site in XA21 using liquid chromatography mass spectrometry and to elucidate the mechanisms underlying the developmental defects observed in lxl1 mutants containing XA21 using dye tracer and anatomical assays. Also using these lxl1 lines, the studies will determine the roles and early molecular events of LXL1-mediated cleavage of XA21 in dehydration responses. Finally, the project will characterize XA21 rice mutants that are insensitive to LXL1 cleavage. This project will significantly advance our understanding of the biology of intramembrane proteolysis in higher plants.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.