Identification Of Interacting Motifs Between ARC1 And Exo70A1 Proteins In Brassica Oleracea

Self-incompatibility(SI) is an important genetic mechanism driving evolution, causing self-pollen rejection and promoting cross-pollination to maintain high heterozygosity, manifesting as pollen tube elongation inhibition during self-pollination. In Brassicaceae, self-incompatibility is regulated by a complex and polymorphic S-locus. This locus produces the S-locus cysteine rich protein(SCR) from self-pollen, which interacts with the stigmatic S-locus receptor kinase(SRK) and promotes signal transduction resulting in pollen rejection. The downstream component of this signal transduction is the armadillo repeat containing 1(ARC1), which is phosphorylated by SRK and located to the cytoplasm. Then, phosphorylated ARC1 combines with Exo70A1 in the cytoplasm, and forms protein complexes mediated by Exo70A1-ubiquitin with the assistance of the ubiquitin-activating enzyme(E1), as well as the ubiquitin-conjugating enzyme(E2). Subsequently, ubiquitinated Exo70A1 moves to a 26 S proteosome/COP9 signalosome(CSN), triggering Exo70A1 degradation, followed by inhibition of pollen germination and pollen tube growth in self-pollinated flowers.In this study, yeast two-hybrid system and GST pull-down technology were used to investigate interaction between ARC1 and Exo70A1 in self-incompatibility Brassica oleracea. The coding sequences of ARC1 and Exo70A1 from eleven Brassica oleracea cultivars were cloned and sequenced. Then bioinformatics analysis were carried out to find the key domain of ARC1 and Exo70A1. Using PCR amplification, site-directed mutation and homologous recombination technology, we obtained several truncated ARC1 segments as well as site-specific mutants with substitutions at possible interaction sites. In addition, Exo70A1 truncation constructs were also generated. Yeast two-hybrid screening was performed to detect interaction between these ARC1 and Exo70A1 constructs, then GST pull-down assay was used to verify the interaction in vitro. Sequencely, the motifs mediated the ARC1-Exo70A1 interaction were identified and subjected to phylogenetic analysis. This work will provide some new insight into the SI signal transduction molecular mechanism.The results are as the followings. 1. Sequence Analysis of ARC1 and Exo70A1A multiple alignment among ARC1 proteins was performed. It revealed a 98.75% identity with a 41-amino-acid-difference. The polymorphic sites are primarily located in the N-terminus of ARC1, however, the U-box domain is highly conserved. The amino acid discrepancy within the ARM sites presents a consistent variety in different cultivars. The multiple alignment result indicates a 99.44% amino acid identity among Exo70A1 proteins with a 24-amino-acid- difference. The Exo70A1 N-terminal domain(1-85aa) only has one difference in residue 38 aa. 2. Truncation and Mutation of Coding Sequence of ARC1 and Exo70A1We analyzed several key domains within ARC1, and four selected truncations were PCR amplified: ARC1210(amino acids 1-210, containing the leucine zipper domain), ARC1246(amino acids 1-246, containing the leucine zipper and coiled-coil domain), ARC1279(amino acids 1-279, containing the UND domain), and ARC1354(amino acids 1-354,containing the UND and U-box domain). Electrophoresis data established that our cloned ARC1 fragments were 630 bp, 738 bp, 837 bp and 1062 bp. Two Ala point-mutants, ARC1354m(amino acids 1-354) and ARC1m(amino acids 1-664), which mutated each of the I272-T277 residues to Ala, were created via overlap-extension PCR. Electrophoresis detection showed that our mutants were 1062 bp and 1992 bp. The encoding sequences of Exo70A1 segments, Exo70A185(amino acids 1-85) and Exo70A1(amino acids 1-638), were PCR amplified, and our Exo70A1 segments were 255 bp and 1919 bp, respectively. 3. Detection of interactions between ARC1 and Exo70A1 derivatives via yeast two-hybrid screeningYeast expression vectors pGADT7-ARC1210, pGADT7-ARC1246, pGADT7-ARC1279, pGADT7-ARC1354, pGADT7-ARC1354 m, pGADT7-ARC1 m, pGBKT7-EXO70A185 and pGBKT7-EXO70A1 were constructed. Toxicity and activation tests indicated that the recombinant plasmids were non-toxic in yeast and the bait plasmids pGBKT7-Exo70A1 cannot auto-activate the reporter. Interaction detection manifested that both ARC1210 and ARC1246 interact strongly with Exo70A185 and Exo70A1, while ARC1279, ARC1354, exhibited a weak interaction. However, ARC1354 m and ARC1 m showed an uncertain and weak interaction with Exo70A1. These results indicated that the N-terminal region of ARC1 is required for efficient binding to the N-terminal region of Exo70A1. 4. Verification of the ARC1 N-terminal Domain as an Interacting Partner with Exo70A1 N-terminal domain via GST Pull-down AssayRecombinant expression plasmids pGEX-4T-1-ARC1210, pGEX-4T-1-ARC1246, PET-15b-Exo70A185 and PET-15b-Exo70A1 were constructed. After optimizing prokaryotic expression conditions under different temperatures and IPTG concentration gradients, we found that pGEX-4T-1-ARC1210, pGEX-4T-1- ARC1246 and PET-15b-Exo70A1 could reach to the highest level when inducing 4h at 32℃ with 0.8mmol/L IPTG, and the molecular weight was 52 kD, 55 kD and 72 kD. While, PET-15b-EXO70A185 was induced overnight at 16℃ with 0.8mmol/L IPTG whose molecular weight was 10 kD. A GST pull-down assay was conducted to further confirm their interaction through the Recombinant GST-tagged ARC1 and His-tagged Exo70A1 using Beaver BeadsTM GSH and Beaver BeadsTM IDA-Nickel. These results indicated that our fusion proteins could interact with each other. 5. Analysis of the ARC1-Exo70A1 Interaction MotifAccording to the results of yeast two hybrid detection and GST pull-down assay, we suggested that the recognition motifs were located within the 1-210 amino acids of ARC1 N-terminal and the 1-85 amino acids of Exo70A1 N-terminal. Aligned with ARC1210 and Exo70A185 in NCBI databases, eighteen ARC1 and twenty-three Exo70A1 sequences from different species were obtained and subjected to construct phylogenetic tree using the MEGA 5.10. Bioinformatic and phylogenetic analysis demonstrated that these motifs were highly conserved across different species, indicating a potential species specificity over the course of evolution. The interaction between these proteins generated a synergistic effect during horizontal evolution in the same species.