FUNCTIONAL STUDIES OF S-RNASE-BASED SELF-INCOMPATIBILITY IN PETUNIA INFLATA

Open Access
- Author:
- Meng, Xiaoying
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 04, 2010
- Committee Members:
- Teh Hui Kao, Dissertation Advisor/Co-Advisor
Teh Hui Kao, Committee Chair/Co-Chair
Hong Ma, Committee Member
Timothy W Mcnellis, Committee Member
Surinder Chopra, Committee Member - Keywords:
- self-incompatibility
Rosaceae
protein degradation
Plantaginaceae
Petunia inflata
Competitive interaction
S-locus F-box protein
Solanaceae
S-RNase
ubiquitination - Abstract:
- Self-incompatibility (SI) is an intraspecific reproductive barrier widely adopted by flowering plants, and it has long been considered responsible for their explosive success. SI allows the pistil to distinguish between genetically related (self) and genetically unrelated (non-self) pollen; self-pollen is rejected, whereas non-self pollen is accepted for fertilization. In the simplest cases, discrimination between self/non-self pollen by the pistil is controlled by a highly polymorphic locus, named the S- locus. Variants of the S-locus are referred to as “haplotypes”. For the Solanaceae type SI, the SI phenotype of pollen is determined by its own S-haplotype; that is, pollen is recognized as self-pollen and rejected by the pistil if its S-haplotype matches one of the S-haplotypes of the pistil. My thesis research focuses on this SI mechanism, and I am using Petunia inflata, a member of the Solanaceae family, as the model plant. The Kao lab has identified the S-RNase gene as the pistil specificity determinant and the PiSLF (P. inflata S-locus F-box) gene as the pollen specificity determinant. The overall goal of my Ph.D. thesis research is to understand how allelic variants of S-RNase and SLF function to elicit S-haplotype-specific inhibition of pollen tube growth in P. inflata. The protein degradation model proposed by the Kao lab suggests that, as a component of an E3 ubiquitin ligase complex, SLF preferentially interacts with its non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome pathway in compatible pollen tubes. In the case of incompatible pollination, S-RNase cannot form stable interaction with its self SLF, thus remains intact and functions as a cytotoxin to inhibit pollen tube growth. Interestingly, many S-locus linked F-box genes have been found in all three families that possess S-RNase-based SI. The Kao lab has identified six such genes. They exhibit S-haplotype-specific RFLP; they are specifically expressed in pollen/pollen tubes; and their deduced amino acid sequences share 50 to 55% sequence identity with PiSLF. In Chapter 2, I describe the use of an in vivo functional assay to examine whether three PiSLF-like genes, PiSLFLb-S2, PiSLFLc-S1 and PiSLFLd-S2, function in SI. This assay is based on the prediction that if a PiSLF-like gene functions in SI, introducing a particular allele of this gene into transgenic plants should cause breakdown of SI function in transgenic pollen carrying a different allele of this gene. I introduced each of these PiSLF-like transgenes into an appropriate S-genotype of wild-type plants, and found that none of them caused breakdown of SI in S3 pollen (in the case of PiSLFLb-S2 and PiSLFLd-S2) or S2 pollen (in the case of PiSLFLc-S1) of the transgenic plants. Although S-RNase is taken up by pollen tubes in a non-S-haplotype-specific manner, it encounters different fates inside self and non-self pollen tubes, as it can exert its cytotoxic activity only in self pollen tubes. In Chapter 3, I describe the study of the fate of S-RNase inside pollen tubes by directly expressing S-RNases in self (incompatible) and non-self (compatible) pollen of transgenic plants. Green fluorescent protein (GFP) was fused at the C-terminus of S-RNase, so that I could monitor the production and localization of S-RNase by examining the GFP fluorescence of in vitro germinated pollen tubes of the transgenic plants. The results suggest that the ectopically produced S-RNase:GFP was not toxic to either its self or non-self pollen. Based on GFP fluorescence of in vitro-germinated pollen tubes, S-RNase:GFP was sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. I also ectopically expressed the non-glycosylated form of S3-RNase, S3-RNase(N29D), in its self and non-self-pollen in transgenic plants, and the results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI. In Chapter 4, I describe functional studies of a truncated PiSLF2 protein, PiSLF2(CTD), which lacks the F-box domain. The transgenic plants expressing PiSLF2(CTD) did not exhibit the same SI behavior as the transgenic plants expressing the full-length PiSLF2, suggesting that the F-box domain is required for the function of PiSLF in SI. This truncated PiSLF2 did not have a dominant-negative effect on the function of the endogenous PiSLF2. In a canonical SCF complex, the F-box protein interacts with Skp1 through its F-box domain. A novel class of Skp1-like protein, named SSK1 (SLF-interacting SKP1-like1), was suggested to be the adaptor to interact with SLF in the SCFSLF complex. I identified the ortholog of SSK1 in P. inflata, named PiSSK1, and examined its interaction with PiSLF1, PiSLF2, and PiSLF3. The results showed that none of the three PiSLFs interacted with PiSSK1. In Chapter 5, I describe several projects I carried out with the aim of using in vivo approaches to determine the roles of PiSBP1 (P. inflata S-RNase Binding Protein 1) and ubiquitination in SI. I tested the hypothesis that PiSBP1 is required for the pollen function in SI as a component of the PiSLF-containing E3 ubiquitin ligase complex and/or as a single subunit E3. I also tested the hypothesis that ubiquitination and degradation of non-self S-RNases in the pollen tube is required for compatible pollination. Although I was unable to draw any definitive conclusion from these experiments, the results obtained will provide useful information for future studies.