Abstract
Plant pathogens secrete effector molecules that suppress the plant immune response to facilitate disease development. AvrPto is a well-studied effector from the phytopathogenic bacterium Pseudomonas syringae. Here we utilize an in planta proximity dependent biotin ligase labeling technique (BioID) in combination with AvrPto to identify proximal proteins that are potential immune system components. The labeling technique biotinylated proteins proximal to AvrPto at the plasma-membrane allowing their isolation and identification by mass spectrometry. Five AvrPto proximal plant proteins (APPs) were identified and their effect on plant immune function and growth was examined in Nicotiana benthamiana leaves. One protein identified, RIN4, is a central immune component previously shown to interact with AvrPto. Two other proteins were identified which form a complex and when silenced significantly reduced P. syringae tabaci growth. The first was a receptor like protein kinase (APK1) which was required for Pto/Prf signaling and the second was Target of Myb1 (TOM1), a membrane associated protein with a phosphatidylinositol 5-phosphate (PtdIns5P) binding motif. We have developed a technology to rapidly determine protein interactions within living plant tissue. It is particularly useful for identifying plant immune system components by defining pathogenic effector protein interactions within their plant hosts.
Highlights
Plants employ receptor kinases (RKs) and receptor proteins as pattern recognition receptors (PRRs) to sense microbes
In order to probe the interactions of AvrPto we designed four constructs which could differentiate between indiscriminate protein interactions at the plasma membrane versus target specific interactions at this interface (Figure 1C)
It is amenable for users to determine the interactions between pathogenic effector proteins and plant host molecules
Summary
Plants employ receptor kinases (RKs) and receptor proteins as pattern recognition receptors (PRRs) to sense microbes. To counter plant defense mechanisms, pathogens utilize cytoplasm targeted effector proteins to alter plant cell function and metabolism to aid infection. Many bacterial effectors target PRRs and their signaling components to prevent immune recognition of the pathogen (Jones and Dangl, 2006). Effector proteins are useful tools for dissecting plant biology due to their abilities to target key. Proteins anchored in a membrane may display strong interactions but when the cell is homogenized and the membrane is no longer acting to stabilize the interface the interaction may be lost. Understanding the spectrum of plant immunity components influenced by effector protein interactions is paramount to identifying solutions for engineering plant disease resistance
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