Competitive yeast three-hybrid assays and non-competitive yeast two-hybrid assays to measure relative strength of protein interactions during the gametophytic self-incompatibility response in Petunia hybrida.

摘要

Gametophytic Self-Incompatibility (GSI) is a specific genetic mechanism that allows plants to recognize and reject "self" pollen, while accepting "non-self" pollen. Two genes, the S-RNase (self-incompatibility ribonuclease) and SLF (S-locus F-box protein) control self/non-self pollen recognition in the pistil and pollen, respectively. Besides these two genes, there are other modifier genes, unlinked to S-locus, are SBP1, SSK1, HT-B, 120K and Cullin1. There are two proposed models for how the GSI system recognizes non-self versus self pollen: (1) SCFSLF F-box/ ubiquitination and (2) Vacuolar sequestration of non-self S-RNases.;My thesis focuses on the first model, which predicts that the interaction of SLF and non-self S-RNase will recruit a SCFSLF E3 ubiquitin ligase complex to the S-RNase leading to its degradation via the 26S proteasome pathway. The main objective of my thesis was to test binding interaction strength between S-RNase alleles/domains and SLF, between S-RNase and SBP1, and between SBP1 and SLF using quantitative yeast two hybrid assays and competitive yeast three hybrid assays.;We confirmed that SBP1 interacted with all different S-RNase domains but not the C-terminal of S-RNase. SBP1 appeared to bind more strongly to S3-NT than S1-NT. SBP1 also showed interaction with the C-terminal of SLF-S1. No interaction of SLF-S1 with different domains of S-RNase was observed on the plate assay. However, weak interaction, apparently stronger for non-self combinations, at least for N-terminal and CHVC domains of S-RNase, was observed between SLF-S1 and S-RNase when fluorescence assays were used. Our result suggests that bridge proteins like SBP1 are required for the formation of stable SLF-S-RNase interaction as proposed by Model I.