| Many plant pathogens, including Pseudomonas syringae, encode the type III secretion system for translocating effector proteins into the host during infection. Strains of P. syringae which are not capable of delivering the type III effectors are nonpathogenic. Therefore, the functions of type III effectors are essential for disease. Although several type III effectors have been demonstrated to block components of the plant defense response, the functions of most type III effectors are unknown. Our lab is interested in the type III effector HopAF1, a type III effector that is present in eleven of the nineteen sequenced strains of P. syringae and other plant pathogens. Although the presence of HopAF1 in multiple strains of P. syringae suggests that it plays an important role in virulence, no function has yet been associated with HopAF1. We generated a tertiary structural prediction for HopAF1, which suggests that HopAF1 is structurally related to bacterial deamidases. Deamidation, the irreversible substitution of an amide group with a carboxylate group, is the mechanism by which several bacterial virulence factors manipulate the activity of a specific substrate. To identify a potential target of HopAF1 activity, we employed a yeast two-hybrid screen and identified Arabidopsis methylthioadenosine nucleosidase (MTN1) as a putative target of HopAF1. This interaction, which we extended to include Arabidopsis MTN2, was confirmed in planta using coimmunoprecipitations and bimolecular fluorescence complementation assays. MTNs are enzymes in the Yang cycle, a cycle essential for high levels of ethylene biosynthesis. Ethylene is a key plant hormone for plant developmental processes, such as senescence and flowering. Ethylene is also induced during PTI. Therefore, we hypothesized that HopAF1 inhibits PTI by manipulating MTNs and levels of ethylene in plants. To this end, we used gas chromatography to measure ethylene biosynthesis in plants treated with bacterially-delivered HopAF1. We determined that HopAF1 inhibits ethylene biosynthesis in a manner dependent on putative catalytic residues. Additionally, Yang cycle mutants mtn1 mtn2 and mtk are more susceptible to weak pathogens. This data is consistent with the idea that HopAF1 targets a novel component of the plant immune system, the Yang cycle. |
