JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2229-2238; doi:10.1093/jxb/eri222
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RESEARCH PAPER |
A coupled yeast signal sequence trap and transient plant expression strategy to identify genes encoding secreted proteins from peach pistils
1Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
2Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea
3Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
* To whom correspondence should be addressed. Fax: +1 607 255 5407. E-mail: jr286{at}cornell.edu
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 self-incompatibility. This pilot YSST screen of pollinated and unpollinated pistil cDNAs revealed a diverse set of predicted cell wall-localized or plasma membrane-bound 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.
Key words: Extracellular proteins, non-S-RNase, peach, pollen–pistil interaction, secreted proteins, yeast signal sequence trap
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