Bacterial pathogens of both plants and humans use a type III secretion system to inject effector proteins into the cells of their hosts. As many as 30 or more different 'type III effectors'can be delivered by an individual pathogen and once inside the host cell they manipulate various processes to promote bacterial virulence. The great diversity of type III effectors, and the fact that their amino acid sequences offer few clues to their function, has been an impediment to understanding how they manipulate the host. One host process, the attachment of ubiquitin to a protein in order to regulate its function or to cause its degradation, plays an important role in host immunity and offers a vulnerable target for type III effectors. This application concerns the characterization of a type III effector, AvrPtoB, from the plant pathogen Pseudomonas syringae. The crystal structure of a C-terminal domain of AvrPtoB revealed that it is a structural mimic of the U-box class of E3 ubiquitin ligases - a type of host protein that facilitates the attachment of ubiquitin to other proteins. The AvrPtoB E3 ligase is able to use host E2 conjugating enzymes to cause ubiquitination and degradation of a host protein kinase required for immunity. Despite these advances, much remains to be learned about how this bacterial E3 ligase binds and ubiquitinates its host substrates in order to subvert their normal functions. Recently virulence proteins from human pathogens such as Legionella pneumophila and Escherichia coli have also been found to express proteins of the U-box class of E3 ligases. Structural mimicry of this type of host protein therefore appears to be a common strategy for undermining host immunity. The long term objective of this project is to increase our understanding of the underlying mechanisms that bacterial E3 ligases use to manipulate host ubiquitination. This knowledge will be useful for the development of novel strategies for interfering with these virulence proteins in order to lessen the impact of pathogen infection in both plants and humans. The goals of this application are to: 1) Identify the host E2 conjugating enzyme used by AvrPtoB and characterize the role of a ubiquitin-interaction motif that lies close to the U-box;2) Exploit the recently solved crystal structure of AvrPtoB in complex with one of its host targets to understand how AvrPtoB recognizes and binds its substrates;3) Perform a screen to identify additional host proteins that AvrPtoB ubiquitinates and which are candidates for playing a role in host immunity;4) Investigate the way in which certain host-mediated modifications of AvrPtoB (e.g. phosphorylation) influence its E3 ligase activity;and 5) Develop a method to visually monitor the delivery of AvrPtoB into the host cell. The method will be used to test the hypothesis that type III effectors that act early in the infection process are delivered before later-acting effectors.

Public Health Relevance

Manipulation of host immunity by a bacterial E3 ubiquitin ligase Gregory B. Martin, Boyce Thompson Institute for Plant Research at Cornell University Project Narrative This research will elucidate how a bacterial virulence protein, which is injected into host cells, interferes with the host immune response. The knowledge gained will contribute to strategies to lessen the impact of bacterial diseases of importance to both agriculture and human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM078021-08
Application #
8600287
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Sledjeski, Darren D
Project Start
2006-05-01
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
8
Fiscal Year
2014
Total Cost
$207,428
Indirect Cost
$49,928
Name
Boyce Thompson Institute for Plant Research
Department
Type
DUNS #
045666088
City
Ithaca
State
NY
Country
United States
Zip Code
14853
Mathieu, Johannes; Schwizer, Simon; Martin, Gregory B (2014) Pto kinase binds two domains of AvrPtoB and its proximity to the effector E3 ligase determines if it evades degradation and activates plant immunity. PLoS Pathog 10:e1004227
Chien, Ching-Fang; Mathieu, Johannes; Hsu, Chun-Hua et al. (2013) Nonhost resistance of tomato to the bean pathogen Pseudomonas syringae pv. syringae B728a is due to a defective E3 ubiquitin ligase domain in avrptobb728a. Mol Plant Microbe Interact 26:387-97
Mural, Ravi V; Liu, Yao; Rosebrock, Tracy R et al. (2013) The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity. Plant Cell 25:3615-31
Clarke, Christopher R; Chinchilla, Delphine; Hind, Sarah R et al. (2013) Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility. New Phytol 200:847-60
Zeng, Lirong; Velasquez, Andre C; Munkvold, Kathy R et al. (2012) A tomato LysM receptor-like kinase promotes immunity and its kinase activity is inhibited by AvrPtoB. Plant J 69:92-103
Cheng, Wei; Munkvold, Kathy R; Gao, Haishan et al. (2011) Structural analysis of Pseudomonas syringae AvrPtoB bound to host BAK1 reveals two similar kinase-interacting domains in a type III Effector. Cell Host Microbe 10:616-26
Munkvold, Kathy R; Martin, Gregory B (2009) Advances in experimental methods for the elucidation of Pseudomonas syringae effector function with a focus on AvrPtoB. Mol Plant Pathol 10:777-93
Dong, Jing; Xiao, Fangming; Fan, Fenxia et al. (2009) Crystal structure of the complex between Pseudomonas effector AvrPtoB and the tomato Pto kinase reveals both a shared and a unique interface compared with AvrPto-Pto. Plant Cell 21:1846-59
Shan, Libo; He, Ping; Li, Jianming et al. (2008) Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity. Cell Host Microbe 4:17-27
Rosebrock, Tracy R; Zeng, Lirong; Brady, Jennifer J et al. (2007) A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity. Nature 448:370-4

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