Characterization of the Interaction Between S-Locus F-Box Proteins and S-Ribonucleases of Petunia inflata Through the Use of Chimeric S-Locus F-box Proteins and CRISPR/Cas9 Knockout

Open Access
Author:
Khatri, Wasi Asif
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Teh-hui Kao, Thesis Supervisor
  • Teh-hui Kao, Honors Advisor
  • Ming Tien, Faculty Reader
Keywords:
  • Petunia inflata
  • CRISPR/Cas9
  • Chimeric Proteins
  • SLF
  • S-RNase
  • petunia
  • self-incompatiblity
  • s-haplotype
Abstract:
Petunia inflata uses a genetic mechanism known as self-incompatibility (SI) to prevent inbreeding and promote outcrossing. SI allows the pistil to reject genetically identical (self) pollen, yet accept genetically dissimilar (non-self) pollen for pollination. Self/non-self recognition is determined by the polymorphic S-locus, which houses the female and male determinant genes. Seventeen SLF proteins (SLF1 to SLF17) have been identified in P. inflata that constitute the male determinant, and a single S-RNase protein constitutes the female determinant. A current model predicts that at least one of the 17 SLF proteins will recognize any non-self S-RNase taken up into a pollen tube to mediate its ubiquitination and degradation, thus resulting in cross-compatible pollination. However, none of the 17 SLF proteins recognize their self S-RNase, allowing the S-RNase to arrest self-pollen tube growth. Although the amino acid sequences of S2-SLF1 (SLF1 of S2-haplotype) and S3-SLF1 (SLF1 of S3-haplotype) are 88.7% identical, S2-SLF1, but not S3-SLF1, interacts with S3-, S7-, and S13-RNases. To determine which domain of the protein is involved in recognition between S2-SLF1 and these three non-self S-RNases, chimeric gene constructs of S2-SLF1 and S3-SLF1 were created and introduced into P. inflata plants via Agrobacterium-mediated transformation. Two chimeric proteins, F322 (containing the first domain of S3-SLF1 and the second and third domains of S2-SLF1) and F232 (containing the first and third domains of S2-SLF1 and the second domain of S3-SLF1) and their interactions with S3-, S6a-, S7-, S12-, and S13-RNases will be explored in this project. Additionally, a current hypothesis predicts that even though there is a suite of SLF proteins that can collectively recognize all non-self S-RNases, there may be some redundancy in the interactions with a particular non-self S-RNase. If each non-self S-RNase were only recognized by one SLF protein, a mutation that abolishes the ability of an SLF protein to interact with the non-self S-RNase it recognizes would result in the pollen being incompatible with normally compatible pistils that produce this non-self S-RNase. Up until this point, S2-SLF1 has been found to interact with the largest number of S-RNases, including S3-, S7-, S12-, and S13-RNases. This hypothesis of redundancy has been explored with the use of the CRISPR/Cas9 genome editing system by knocking out S2-SLF1 in S2S3 plants and using that plant to pollinate the pistils of various other S-haplotype. It was found that S2 pollen lacking S2-SLF1 was still compatible with S7S7 plants, suggesting at least one other SLF protein is able to recognize S7-RNase and mediate its ubiquitination and degradation. This project uses these findings to examine the interactions of S2-SLF2, S2-SLF12, S2-SLF14, and S2-SLF16 with S7-RNase.