Protein Kinase Signaling Cascades Regulating Plant Cell Death and Their Manipulation During Plant-pathogen Interactions

Diseases of crop plants continue to pose major economic and environmental challenges to U.S. agriculture. As one example relevant to this project, growers of fresh market tomatoes often apply copper-based pesticides 10-12 times per season in order to control bacterial speck disease. These applications incur not only an economic cost in the form of labor, machinery, petroleum fuels, and pesticides but also an environmental cost because much of the pesticide ends up in non-target environments such as local water supplies. The development of plants that are more resistant to diseases will reduce our dependence on chemical pesticides, produce economic benefits for the farmer, and provide food for U.S. consumers that has fewer pesticide residues. These goals can be best achieved through an understanding of the molecular basis of the interaction of plants with their pathogens in order to genetically improve plant traits for disease resistance. The interaction of the bacteria Pseudomonas
syringae pv. tomato (Pst) with tomato is both economically important and a model system for studying the molecular basis of disease resistance and susceptibility in plants. In susceptible tomato plants, infection by Pst causes bacterial speck disease. This disease is characterized by small (1mm) necrotic lesions ('specks') that become surrounded by chlorotic haloes. Disease symptoms also form on tomato fruits and these blemishes, combined with lower yields due to loss of leaves, cause serious economic losses to farmers throughout the world. In addition to its economic significance, the tomato-Pst interaction offers many experimental advantages and thus it has emerged as a model system to understand the molecular basis of pathogen-host interactions for both resistance and disease susceptibility. Both tomato and Pst are experimentally tractable. Tomato is diploid, has a relatively short life-cycle, tolerates inbreeding yet is easily cross-hybridized, and is simple to grow and
maintain. Many germplasm resources are available for pathogen interaction studies including Pst resistant plants termed PtoR and Pst susceptible plants termed PtoS and prf-3. Also, many techniques are available for tomato including routine transformation and virus-induced gene silencing (VIGS). A genome sequence of tomato is also available. Pst is also amenable to molecular genetic analysis and is the subject of a project that generated the full genome sequence for the DC3000 strains, a draft sequence of the T1 strain (9), and a near-complete inventory of effector proteins delivered by the type III secretion system. Resistance to Pst is based on a "gene-for-gene" interaction involving the tomato Pto kinase and the Pst AvrPto and AvrPtoB effector proteins. Tomato plants expressing Pto (PtoR plants) recognize Pst strains which express either of two effector proteins, AvrPto or AvrPtoB. These interactions result in resistance to bacterial speck. Pto was identified in the 1980s and
currently provides control of bacterial speck disease on ~60% of tomato acreage in California. The Pto gene was cloned in 1993 and found to be a member of a clustered gene family. Programmed cell death (PCD) is an essential process for development and immune responses in eukaryotic multicellular organisms. In mammalian systems, PCD is used for many processes from digit formation to removal of infected cells. In plants, leaf senescence, development of xylem tracheary elements, and host responses to pathogens all involve PCD. Localized PCD occurs in both resistant and susceptible plants during pathogen attack. In resistance, PCD termed the hypersensitive response (HR) occurs rapidly (<12 hr) upon pathogen recognition and is thought to limit pathogen spread to the site of infection. The interaction of Pto with AvrPto or AvrPtoB leads to the HR and resistance in tomato. In disease-susceptible plants, localized cell death referred to as 'specks', 'spots', or
'blights' appears over the course of many days. Studies have suggested that these disease symptoms also involve host-mediated PCD. Thus, host-controlled PCD plays a critical role in determining both immunity and disease progression in plants. Despite this significance, relatively few plant genes have been identified which have a demonstrated role in PCD associated with pathogen attack and upstream components that might regulate these cell death mediators are yet to be identified. Identification of genes involved in plant programmed cell death has proven difficult. While many processes in plants that require PCD are known, identification of genes that play a direct role in plant PCD signaling pathways has been elusive compared to mammalian systems. However, in recent years, the number of genes identified to be involved in plant PCD control has increased and include the protein kinase we study, Adi3, BAG proteins, Bax inhibitor-1, lipid biosynthesis genes, MAP kinases, and
ubiquitin E3 ligases. The tomato protein kinase Adi3 interacts with Pto and AvrPto and functions as a negative regulator of host PCD. A yeast three-hybrid (Y3H) system was developed in an effort to identify tomato proteins that interact with Pto only in the presence of AvrPto and thus, may play a role in resistance.These proteins were termed Adi proteins (AvrPto-dependent Pto-interacting proteins). We have characterized the Adi3 protein and shown that it interacts with Pto only when co-expressed with AvrPto in the Y3H system. We used a combination of reverse genetic, molecular, and biochemical techniques to characterize Adi3 as a negative regulator of host PCD connected with pathogen-associated cell death. Adi3 is an AGC protein kinase activated when phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). Adi3 belongs to group VIIIa of plant AGC (for PKA, PKG, and PKC) protein kinases (33). This group is characterized by a large 80 to 100 amino acid extension to the
domain known as the T-loop (or activation loop) that is phosphorylated for kinase activation. This plant specific T-loop extension appears to contain cellular localization signals that may be important for kinase function. Recently, we have shown that nuclear localization is required for Adi3 cell death suppression under non-pathogen conditions. Mutation of the nuclear import signal prevents Adi3 nuclear entry, accumulates Aid3 in endosomal vesicles, and induces PCD through a loss of Adi3 cell death suppression. Taken together, our data suggest that Adi3 functions in a signaling pathway to suppress PCD under non-pathogen challenged situations. In the presence of AvrPto and Pto, Adi3 cell death suppression is released by prevention of Adi3 nuclear entry, which restricts Adi3 to endosomal vesicles and brings about host HR-like PCD for resistance. This proposal will focus on the mechanisms by which Adi3 suppresses cell death in the absence of Pst, and how the tomato-Pst interaction
brings about the inactivation of Adi3 cell death suppression during resistance. Additionally, this proposal will include studies on a recent finding in the Devarenne lab that the enzyme threonine deaminase is regulated in response to Pst to control the production of the defense hormone jasmonic acid.Objectives: In an effort to better understand the genes and pathways involved in the regulation of plant cell death and how these pathways are manipulated during the resistance response to Pst, our research will focus on:1. Investigating the role of the SnRK complex in the defense response to Pst.2. Analyzing nuclear phosphorylation events related to PCD that are controlled by Adi3.3. Examining alterations in nuclear Adi3 PCD-related phosphorylation during Pst resistance.