S-ribonuclease (S-RNase)-based self-incompatibility (SI) mechanisms have been extensively studied in Solanaceae, Rosaceae and Plantaginaceae. S-RNase-based SI is controlled by two closely related genes, S-RNase and S-locus F-box (SLF), located at a polymorphic S-locus. In the SI system, the SCF-type (SKP1-CUL1-F-box-RBX1) complex functions as an E3 ubiquitin ligase complex for ubiquitination of non-self S-RNase. Pummelo (Citrus grandis) and several mandarin cultivars are suggested to utilize an S-RNase-based SI system. However, the molecular mechanism of the non-S-factors involved in the SI reaction is not straightforward in Citrus. To investigate the SCF-type E3 complex responsible for the SI reaction in mandarin, SLF, SKP1-like and CUL1 candidates potentially involved in the SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco) were identified based on the genome-wide identification and expression analyses. Sixteen pollen-specific F-box genes (CrFBX1-CrFBX16), one pollen-specific SKP1-like gene (CrSKP1-e) and two CUL1 genes (CrCUL1A and CrCUL1B) were identified and cloned from ‘Wuzishatangju’. Yeast two-hybrid (Y2H) and in vitro binding assays showed that five CrFBX proteins could bind to CrSKP1-e, which is an ortholog of SSK1 (SLF-interacting-SKP1-like), a non-S-factor responsible for the SI reaction. Luciferase complementation imaging (LCI) and in vitro binding assays also showed that CrSKP1-e interacts with the N-terminal region of both CrCUL1A and CrCUL1B. These results indicate that CrSKP1-e may serve as a functional member of the SCF-type E3 ubiquitin ligase complex in ‘Wuzishatangju’.
Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays
