A coupled yeast signal sequence trap and transient plant expression strategy to identify genes encoding secreted proteins from peach pistils

Abstract

Many developmental processes and induced plant responses have been identified that are directly or indirectly influenced by wall-localized, or apoplastic, molecular interactions and signalling pathways. The yeast-based signal sequence trap (YSST) is a potentially valuable experimental tool to characterize the proteome of the wall and apoplast, or 'secretome', although few studies have been performed with plants and to date this strategy has not been coupled with a subsequent analysis to confirm extracellular localization of candidate proteins in planta. This current report describes the use of the YSST, together with transient expression of a selection of identified proteins as fusions with the reporter GFP, focusing on the complex extracellular interactions between peach (Prunus persica) pollen and pistil tissues. The coupled YSST and GFP localization assay was also used to confirm the extracellular localization of a recently identified pistil-specific basic RNase protein (PA1), as has been observed with S-RNases that are involved in selfincompatibility. This pilot YSST screen of pollinated and unpollinated pistil cDNAs revealed a diverse set of predicted cell wall-localized or plasma membranebound proteins, several of which have not previously been described. Transient GFP-fusion assays and RNA gel blot analyses were used to confirm their subcellular localization and to provide further insights into their expression or regulation, respectively. These results demonstrated that the YSST strategy represents an effective means either to confirm the extracellular localization of a specific candidate secreted protein, as demonstrated here with PA1, or to conduct a screen for new extracellular proteins.

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

“…GFP was exported from the cells and accumulated in the apoplast when the secretory leader was attached to GFP ( Figure 3A) but accumulated in the cytoplasm and nucleus when the leader was not attached ( Figure 3B), as has been observed by others (e.g., Bonello et al, 2002;Yamane et al, 2005). When fulllength Avr1b was fused to GFP, the proteins also accumulated in the apoplast if a mutation was present in either the RXLR2 motif ( Figure 3C) or in the dEER motif ( Figure 3D).…”

An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens

“…Plasma membrane labeling was based on the full-length coding region of At-PIP2A, a plasma membrane aquaporin, acquired from The Arabidopsis Information Resource Stock Center (ABRC stock numbers: CD3-1003; Cutler et al, 2000;Nelson et al, 2007) fused to the N terminus of GFP. Approximately 1 mg each of the PpABCG7-RFP and AtPIP2A-GFP plasmids was mixed and used to bombard onion (Allium cepa) epidermal cells as described by Yamane et al (2005). The tissue was incubated for 16 to 24 h at 24°C in the dark, the epidermal layer was peeled, transferred to glass slides, and mounted in a 30% Suc solution to induce plasmolysis, and the cells were observed using a Leica TCS-SP5 confocal scanning laser microscope (Leica Microsystems).…”

The linkage between cell wall metabolism and fruit softening: looking to the future

“…This is a challenging objective, since the isolation of cell wall proteins is notoriously difficult, but several new tools have been developed, including techniques to isolate highly purified wall protein extracts, functional screens for secreted proteins, and bioinformatic analyses, that collectively hold great promise. 83,84 For example, a preliminary analysis of the tomato fruit cell wall proteome, or 'secretome', using a yeast secretion trap screen (described by Yamane et al 85 ) revealed that approximately 40% of the identified secreted proteins could be classified as 'new' cell wall proteins. This designation meant that either the protein function was entirely unknown, since there was no revealing sequence homology to a functionally characterised protein, or that the location of the annotated protein was unanticipated, based on its predicted function (Lee H and Rose JKC, unpublished).…”