Development of a Rapid in planta BioID System as a Probe for Plasma Membrane-Associated Immunity Proteins

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.

Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID

“…Time course experiments in N. benthamiana suggest that miniTID is equally fast, with clear labeling of proteins visible within 10 minutes (Figure 1figure supplement 3). This is a significant improvement over BirA*, for which labeling times of 24 hours were applied in all three published plant experiments (Khan et al 2018;Conlan et al 2018;Lin et al 2017).…”

“…The inclusion of a biotindepletion step was found to be critical as free biotin in the protein extracts competes with biotinylated proteins for binding of the streptavidin beads (Figure 4figure supplement 6). While for mammalian cell culture or rice protoplasts thorough washing of the cells seems to suffice for removal of free biotin, this is not the case for intact plant tissue (see also (Conlan et al 2018;Khan et al 2018)). Especially when large amounts of starting material and moderate amounts of biotin are used, little to none of the biotinylated proteins may be bound by the beads.…”

“…Although the potential utility for PL in plants is equally tremendous, its adaptation in plants is proving challenging. So far, only three studies have described successful PL in plant systems, employing overexpression of a transcription factor (TF) in transiently transformed rice protoplasts (Lin et al 2017) and high level expression of Pseudomonas syringae effector proteins in stable Arabidopsis lines (Khan et al 2018) and transiently transformed N. benthamiana leaves (Conlan et al 2018), respectively.…”

Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

“…Next, we tested whether we could achieve biotinylation of protein interactors using PDL under the conditions established for N. benthamiana . We observed that the bait proteins used in plants for PDL so far were either membrane-anchored and small proteins [HopF2 (Khan et al, 2018) and AvrPto (Conlan et al, 2018)], or nuclear and/or cytoplasmic localized [OsFD2 (Lin et al, 2017), N (Zhang et al, 2019) and FAMA (Mair et al, 2019)].…”

“…In plants, the number of reports on the use of PBLs is slowly increasing. So far, four papers describe the application of the first generation of PDLs in plants (Conlan et al, 2018; Das et al, 2019; Khan et al, 2018; Lin et al, 2017). In these first trials, overexpression of BioID was combined with long labelling times, very high biotin levels and relatively poor labelling efficiencies.…”