Protein interactions in gametophytic self-incompatibility: Bimolecular fluorescence complementation assays in Petunia hybrida and data-mining for SLF-variant sequences in Petunia inflata.

摘要

Gametophytic self-incompatibility in Petunia hybrida is an outbreeding mechanism in which "self" pollen is recognized and rejected. The two S-locus linked recognition components in this response are the S-RNase (self-incompatibility ribonuclease, Pistil-S) and SLF (S-locus F-box protein, Pollen-S). Additional proteins, not linked to the S-locus, including SSK1, HT-B, 120 kDa, and SBP1, also participate in the rejection process. SLF, S-RNase, SBP1 and other proteins have been shown to interact in vitro by yeast two-hybrid and pull-down assays. We undertook experiments to determine if SLF, S-RNase and other proteins thought to be involved in self-incompatibility interactions can interact in vivo. For these experiments, we used bimolecular fluorescence complementation (BiFC), a technique that allows the interaction and localization of proteins to be visualized in vivo. SLF, S-RNase and SBP1 proteins were fused to N- or C-terminal subfragments of Venus and SCFP3A fluorescent proteins using Gateway(TM) cloning. For transient-expression interaction assays, these constructs were co-transformed into leaf protoplasts and introduced into Nicotiana benthamiana leaf tissue using agroinfiltration with Agrobacterium tumefaciens. Fluorescence resulting from protein interactions in vivo was assayed by confocal microscopy. We were able to see strong interactions between all different S-RNase domains and SBP1. The SBP1 protein also interacted with the SLF-S1 protein. However, binding between SLF-S1 and different alleles/domains of the S-RNase protein appeared to be very weak.;The bioinformatics portion of this thesis focused on identifying gene numbers and characterizing coding sequences for SLF and S-RNase genes in the Petunia inflata genome sequence database. The sequences for known variants of SLF and known S-RNase alleles were used in BLASTn searches to identify scaffolds with homology to the different genes. Seven of eight SLF variants were present in Petunia inflata, and appeared to have intact coding regions that will produce a functional SLF protein. One SLF variant, SLF7 appeared to be reaarranged and is therefore a pseudogene in this line. The S-RNase gene identified had close similarity with the S3-RNase of Petunia hybrida, and had an intron/exon structure identical to that of previously characterized S-RNase alleles. Because the sequence differences between the P. inflata S-RNase and the P. hybrida S3-RNase were concentrated in the hypervariable domains, this may represent a new S-RNase allele.