Functional studies of S-RNase-based self-incompatibility in Petunia inflata

Open Access
Author:
Sun, Penglin
Graduate Program:
Plant Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 19, 2015
Committee Members:
  • Teh Hui Kao, Dissertation Advisor
  • Teh Hui Kao, Committee Chair
  • Michael Axtell, Committee Member
  • Dawn S Luthe, Committee Member
  • Timothy W Mcnellis, Committee Member
Keywords:
  • Petunia inflata
  • self-incompatibility
  • S-RNase
  • S-locus F-box protein
  • ubiquitin-26S proteasome pathway
  • artificial microRNA
Abstract:
Many flowering plants possess self-incompatibility (SI), an intraspecific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote outcrossing. In Petunia, the polymorphic S-locus, which controls self/non-self recognition, contains an S-RNase gene which regulates pistil specificity and multiple S-locus F-box (SLF) genes which collectively regulate pollen specificity. The collaborative non-self recognition model predicts that, for a given S-haplotype, each SLF recognizes a subset of non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, and multiple SLFs collaboratively recognize and detoxify all non-self S-RNases to allow compatible pollination. The overall goal of my dissertation research is to study how SLF proteins interact with S-RNases in order to understand the biochemical basis of self/non-self recognition during SI interactions. The function of the S-RNase gene in controlling pistil specificity has been established through both gain- and loss-of-function experiments; however, only gain-of-function experiments have been used to demonstrate the function of several types of SLF genes in controlling pollen specificity. In Chapter 2, using a gain-of-function assay, I examined the relationships between S2-SLF1 (S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell specific promoter, Late Anther Tomato 52 (LAT52), suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases, and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF. An SLF is predicted to be the F-box protein component of an SCF complex (composed of Cullin1, Skp1, RBX1 and an F-box protein), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. In Chapter 3, I used pollen extracts of a transgenic plant expressing S2-SLF1:GFP for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). I identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an RBX1). Thus, all components but RBX1 of the SLF-containing complex may have evolved specific isoforms in SI. According to the collaborative non-self recognition model, SLFs collectively mediate ubiquitination and degradation of non-self S-RNases. In Chapter 4, I describe a surprising finding that SLF1 of P. inflata itself was subject to degradation via the ubiquitin-26S proteasome pathway (UPP), and identified an 18-amino-acid (18-aa) sequence in the C-terminal domain of S2-SLF1 (SLF1 of S2-haplotype) that contains a degradation motif. Four of the 18 amino acids are conserved among all 17 SLF proteins of S2-haplotype and S3-haplotype involved in pollen specificity, suggesting that the stability of all SLF proteins is likely subject to similar regulation. Deleting the 18-aa sequence from S2-SLF1 stabilized the protein but abolished its function in SI, suggesting that regulation of the stability of SLF proteins is an integral part of their function in SI. I used S2-SLF1:GFP as the bait to perform Co-IP followed by MS, and identified PiDCN1 (defective in cullin neddylation 1) and PiUBC12 (NEDD8-conjugating enzyme) that may be involved in neddylation of PiCUL1-P to activate SCFSLF complexes; and identified PiCAND1 (cullin-associated NEDD8-dissociated protein 1), UPL1-like protein (a mono-subunit E3 ligase containing a HECT domain) and an E2 ubiquitin-conjugating enzyme, that may mediate dissociation of SLF proteins from their SCFSLF complexes and degradation. In Chapter 5, I describe an ongoing project to validate the role of PiDCN1 and PiUBC12 in neddylation of PiCUL1-P, and the function of PiCAND1 in dissociation of SLF proteins from their SCFSLF complexes, based on the results obtained from Chapter 4. I also discuss some of the experiments I carried out to further pinpoint the degradation motif of S2-SLF1 in yeast. Finally, in Chapter 6, I summarize all the major findings of my dissertation research, and describe how these findings have advanced our understanding and laid a foundation for future studies of S-RNase-based SI.