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INVESTIGATOR: Frame, K.

PERFORMING INSTITUTION:
STANFORD UNIV
STANFORD, CALIFORNIA 94305

ROLE OF LIPID SIGNALING IN PLANT RESISTANCE TO BACTERIAL PATHOGENS EXPRESSING AVRBST

NON-TECHNICAL SUMMARY: As global food demand increases, farmers will have to keep food prices competitive while dealing with increasingly costly agricultural inputs. One way to achieve this goal is to reduce the amount of crops lost to pathogens before even being harvested. The goal of this research project is to understand how the pathogen Xanthomonas campestris pathovar vesicatoria (Xcv) causes bacterial spot disease, a commercially important disease of tomato and pepper. Xcv, as well as other bacterial pathogens of plants and animals, has evolved a syringe-like apparatus called the Type 3 Secretion System (T3SS) to infect host cells. The pathogen uses this apparatus to inject so-called effector proteins into plant cells to deactivate host defense systems in order to promote pathogen growth and dispersal. This research project will focus on the functional characterization of the Xcv effector, AvrBsT. We will determine how AvrBsT directly affects the plant innate immune system by determining the key cellular components that AvrBsT is targeting, and how the plant cell responds to such perturbation. AvrBsT is a member of a specific family of T3SS effectors shared widely by plant pathogens (e.g. Ralstonia and Pseudomonas) and animal pathogens (e.g. Yersinia and Salmonella). This research will thus not only uncover mechanisms for how Xanthomonas overcomes plant defenses, it will also provide fundamental insight into how diverse hosts may have evolved immunity against these virulence factors. Such work can be used to prevent or eliminate infection in plant and animal systems, which will benefit crop quality, production and human health.

OBJECTIVES: The bacterial pathogen Xanthomonas campestris vesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Strains carrying AvrBsT, an effector protein injected into plant cells by the Type 3 Secretion System (T3SS), can colonize only tomato cultivars, whereas all known pepper cultivars are resistant. The Arabidopsis thaliana - Pseudomonas syringae model pathosystem was exploited to investigate the genetic and biochemical mechanisms by which AvrBsT suppresses and activates host immune responses in susceptible and resistant ecotypes, respectively. It was discovered that plants lacking SOBER1 phospholipase enzymatic activity are resistant to Pseudomonas expressing AvrBsT, and that resistance is correlated with a burst of the defense signaling molecule phosphatidic acid (PA). Further work suggests that the putative apolipoprotein CIP may be an important regulator of plant innate immunity and lipid dynamics. This proposal will investigate how CIP affects lipid-mediated defense signaling, how CIP-AvrBsT interactions modulate host resistance, and identify additional regulators of plant immunity. Objective 1: Characterize Arabidopsis CIP and determine how it alters AvrBsT-elicited immunity during bacterial infection. We have discovered that CIP binds weakly to SOBER1 and strongly to AvrBsT. CIP overexpression appears to enhance host resistance, suggesting that CIP may be a positive regulator of AvrBsT-elicited immunity. This aim will characterize the role of CIP in plant immunity by determining: (1) if CIP directly binds phospholipids affecting PA levels, phospholipid metabolism and/or flux and (2) if CIP directly or indirectly regulates phospholipase D activity. Objective 2: Characterize CIP localization and post-translational modification during AvrBsT-elicited immunity. Microscopy studies suggest that CIP is a microtubule-associated protein. CIP interacts with AvrBsT in yeast and in pull-down assays in vitro. These results suggest that AvrBsT may target CIP and/or microtubule dynamics. The enzymatic activity of AvrBsT is unknown; however, AvrBsT catalytic mutants are unable to elicit defense responses in resistant plant lines. The characterized enzymatic activities of its family members suggest that AvrBsT may be an acetyltransferase, a sumo protease and/or an ubiquitin protease. This suggests that AvrBsT-dependent post-translational modification of CIP might alter defense signaling in plants. This aim will: (1) determine CIP subcellular localization changes in plant cells due to AvrBsT and (2) determine if AvrBsT post-translationally modifies CIP. Objective 3: Perform a genetic screen to identify regulators of AvrBsT-elicited immune responses. The identification of two regulators (SOBER1 and CIP) of AvrBsT-elicited immune responses suggests that plant cells carefully regulate lipid signals during infection to control the magnitude and/or duration of defense signaling. However, additional genes appear to regulate phospholipid levels during AvrBsT-elicited immunity. This aim will develop tools to study AvrBsT-dependent PA responses and use them to perform a genetic screen to isolate genes regulating this pathway.

APPROACH: Objective 1: Characterizing Arabidopsis CIP and determine how it alters AvrBsT-elicited immunity during bacterial infection. In this aim, various mechanisms by which CIP may alter AvrBsT-elicited immunity or phospholipid dynamics will be explored. CIP-phospholipid binding will be assayed by incubating recombinant CIP protein with commercially available phospholipids and assaying binding by chemiluminescence using anti-CIP antibodies. The Phospholipase D (PLD) isoforms responsible for the phosphatidic acid burst which correlates with AvrBsT-triggered immunity in Arabidopsis will be determined by assaying AvrBsT resistance of PLD mutants in a resistant background. The ability of CIP to bind these isoforms and/or alter their activity will then be assayed via Yeast Two-Hybrid and in vitro pulldowns, as well as in vitro enzymatic assays. In addition, CIP knockout mutants will be phenotyped for differences in immunity during AvrBsT infection and phospholipid levels. Objective 2: Characterizing CIP localization and post-translational modification during AvrBsT-elicited immunity. The effect of AvrBsT on CIP localization will be assayed by introducing a dexamethasone inducible AvrBsT construct (DEX::AvrBsT) into Arabidopsis stably expressing labeled CIP under its own promoter (ProCIP::GFP-CIP) or into Arabidopsis coexpressing CIP and a microtubule marker (ProCIP::GFP-CIP/CHERRY-TUA5). AvrBsT expression in these lines will then be induced with dexamethasone and changes in CIP or microtubule localization monitored. CIP posttranslational modification will be assayed by infiltrating ProCIP::GFP-CIP Arabidopsis with Pseudomonas expressing AvrBsT and immunoprecipating the GFP-CIP from the plants at 0, 3 and 6 hours post inoculation. Ubiquitin, SUMO, and anti-acetylated lysine antibodies will then be used to monitor changes in CIP posttranslational modification. Direct enzymatic activity of AvrBsT upon CIP will then be studied in enzymatic assays in vitro. Objective 3: A genetic screen to identify negative regulators of AvrBsT-elicited immune responses. As AvrBsT-dependent resistance was not conferred on Col-0 sober1-3 null mutants, it is likely that additional negative regulators of resistance exist in this background. These regulators will be identified with a genetic screen. 20,000 homozygous Col-0 sober1-3 plants will be EMS mutagenized to induce random point mutations. Mutants will be screened by HR testing as in Aim 1 or by bacterial dipping. Mutants that undergo HR or display decreased chlorosis and necrosis when inoculated with Pseudomonas expressing AvrBsT have likely undergone a loss-of-function mutation. Mutants will be backcrossed into the parental genotype, and complementation groups identified. Interesting mutants will be crossed into Pi-O and the mutated gene identified by map-based cloning. Further characterization will include bacterial growth curves and genetic complementation assays. As this screen is only expected to find negative regulators of AvrBsT-dependent resistance, it may be necessary to increase the screening population in order to saturate it.