Biochemical Recognition Specificity and Evolution of S-locus F-box Genes in Petunia Based Self-Incompatibility
Open Access
- Author:
- Williams, Justin Stephen
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 25, 2016
- Committee Members:
- Dr. Teh-hui Kao, Dissertation Advisor/Co-Advisor
Dr. Teh-hui Kao, Committee Chair/Co-Chair
Dr. Wendy Hanna-Rose, Committee Member
Dr. Ross C. Hardison, Committee Member
Dr. Claude W. dePamphilis, Outside Member - Keywords:
- self-incompatibility
petunia
SI
SLF
transcriptome
self/non-self recognition
solanaceae
genome assembly
S-ribonuclease
S-RNase
S-locus F-box
F-box - Abstract:
- Deleterious effects from inbreeding can arise through recessive mutations and is observed in many organisms, including plants. The immobility of plants and the physical proximity of male and female organs can result in a propensity for inbreeding, making it especially crucial for bisexual plants to develop strategies to prevent this from occurring. Self-incompatibility (SI) is an interspecific reproductive barrier which allows flowering plants to recognize and reject self-pollen (genetically identical) and accept non-self pollen (genetically distinct). Using Petunia inflata (Solanaceae) as a model, this distinction between self vs non-self pollen is determined by the highly polymorphic S-locus; if the S-haplotype (denoted S1, S2, S3, etc.) of the pollen is identical to either of the S-haplotypes of the diploid pistil, this pollen is rejected. The S-locus houses both female and male determinant genes responsible for this specificity. The female determinant gene, encoded by a single S-Ribonuclease gene (S-RNase), is specifically expressed in the transmitting cells of the pistil. S-RNase is taken up by both self-pollen and non-self pollen, after pollen tubes have penetrated the stigma, arresting self-pollen tube growth in the upper two-thirds of the style; the cytotoxic effect of S-RNase in self-pollen is more than likely due to the degradation of cytosolic RNAs. In stark contrast, the male determinant genes are encoded by multiple pollen-specific S-locus F-box genes (named SLF1, SLF2 … SLF17). Like other F-box proteins, Petunia SLFs are members of a conventional E3-ubiquitin ligase complex, termed the SCF complex. In the conventional SCF complex, the F-box confers target specificity for the polyubiquitination of a substrate protein, marking it for degradation via the 26S proteasome pathway. The SCFSLF complex is thought to work in a similar way, with each SCFSLF acting to collaboratively interact with and ultimately detoxify, all non-self S-RNases as the substrate proteins. Achieving an in-depth understanding of self-incompatibility, means discerning the biochemical basis for these SLF/non-self S-RNase interactions. At the start of my dissertation research, only 10 paralogous SLF genes had been discovered in Petunia, however the total number of SLF genes required for pollen specificity was unknown. Before being able to characterize the interaction relationships between SLF/non-self S-RNases, the total suite of SLF genes involved in SI must first be known. Historically, many methods have been used to incrementally discover new SLF genes, however a more comprehensive method was desired. Using a pollen-specific next-generation sequencing approach, the transcriptomes of S2 pollen, S3 pollen, and S3S3 leaf (as a control) were analyzed. Chapter 2 describes the results of this transcriptomic approach which lead to the discovery of seven additional SLFs in both S2 pollen, S3 pollen, raising the total number of paralogs of SLF to 17. Determining the biochemical basis of SLF/non-self S-RNase interactions is predicated not only upon knowing all types of SLF, but also which SLFs interact with which non-self S-RNase(s). Additionally, by determining the interaction specificity of a given SLF confirms its role in SI. To determine these interactions, a well defined in vivo transgenic approach is used. For example, if S2-SLF1 expressed in S2S3 transgenic plants causes the breakdown of SI, this shows us that S2-SLF1 must breakdown SI in S3 pollen, meaning S2-SLF1 interacts with the non-self S3-RNase. Chapter 3 describes the results of using this in vivo transgenic approach to test the non-self S-RNase interaction specificity of S3-SLF1, S3-SLF5, S3-SLF6, S3-SLF9, S3-SLF10, S2-SLF2, S2-SLF7, S2-SLF9, S2-SLF12, S2-SLF14, S2-SLF16, and S2-SLF17 in SI. Previous unpublished work has indicated that the difference of interaction specificity between S2-SLF1 and S3-SLF1 with S3-RNase is in the last of three functional domains (FDs), termed FD3. These three FDs were delineated by equally dividing the length of SLF1 into thirds, placing the F-box domain in FD1, and the SLF C-terminal domain (CTD) in FD2 and FD3. The role of FD3 was ascertained by generating a series of chimeric SLF1 genes by swapping FD1, FD2, and FD3, and observing the breakdown of SI via in vivo transgenic functional assays. Based upon alignments between S2-SLF1 and S3-SLF1, there are a total of sixteen amino acid differences in FD3. To narrow down the amino acid positions responsible for this difference of interaction specificity, FD3 was further divided into four mini-domains each containing four of the sixteen amino acid differences, termed FD3-A, FD3-B, FD3-C and FD3-D. In another series of chimeric experiments by constructing five chimeric genes between S2-SLF1 and S3-SLF1 via domain swapping, the number of amino acid positions involved in this differential interaction was reduced to eight, shown in Chapter 4. Additional testing of the original FD1, FD2, and FD3 chimeric SLF1 proteins also indicates that these domains interact in a similar manner with non-self S-RNases other than S3-RNase. Chapter 5 describes another experiment aimed at further understanding SI by sequencing the S2-locus, which until recently, has for the most part only been described genetically. Using the sequences of 17 SLF genes to screen the S2-haplotype BAC library (Bacterial Artificial Chromosome), we can identify large portions of genomic DNA which contain these SLF genes. In collaboration with another graduate student, Lihua Wu, we have isolated and purified these SLF-containing BACs, as well as BACs from a previously identified 881-Kbp region surrounding the female determinant gene S2-RNase, and male determinant gene S2-SLF1. Using a hybrid approach by combining two different sequencing technologies (PacBio SMRT and Illumina MiSeq sequencing), I have successfully designed a pipeline to leverage the long, yet error-prone reads from PacBio and the relatively short, yet high-quality reads of MiSeq (combining software programs ABySS and AHA) into scaffolds containing these 17 SLF genes. Thus far, over 3 Mbp of sequence data has been assembled, and analyzed. In summarization of these results, Chapter 6 details the main findings of my dissertation research, including the discovery of seven additional types of SLF (SLF11-17) in five different S-haplotypes (totaling 42 novel genes), and the molecular characterization of the S2-locus by sequencing and assembly of SLF-containing BACs. These findings raise interesting questions about the development of Petunia SI; these questions and new avenues of research for further understanding of SI are discussed.