Study On SCR S Haplotype-dependent Interaction With Chimeras And Mutants Of ESRK In Brassica Oleracea L. Var.Capitata L.

The selective pressure on hermaphrodite flowering plants to prevent inbreeding has been a powerful evolutionary driving force, resulting in the evolution of numerous self-incompatibility (SI) systems. SI is a model system to study plant cell signaling. Amongst members of the Brassicaceae, SI is characterized by the inhibition of self-or self-related pollen at the stigmatic surface. The molecular events of Brassica SI have been elucidated in some detail and depend on a receptor:ligand interaction that occurs at the stigmatic surface. The receptor is a membrane-spanning serine/threonine receptor kinase termed S-locus receptor kinase (SRK). SRK is located in the plasma membrane of the papillar cells that cover the stigmatic surface. The extracellular domain (eSRK), which is responsible for ligand binding, is highly polymorphic between haplotypes, as one would expect for a specificity determining molecule. The ligand for SRK is termed S cysteine-rich (SCR) and has also been designated SP11. SCR is a member of one family of small, cysteine-rich proteins. The haplotype specific binding of SCR to SRK result in rejection of self-pollen.To date, the specificity determinants of SRK have not been examined experimentally; however, bioinformatic speculation has resulted in a number of hypotheses as to which region(s) of the molecule is involved. It is known that eSRK is responsible for ligand binding and eSRK can be split into three subdomains based on sequence similarity. The N-terminal region is similar to that of mannose-binding lectins, the middle hyper variable region contains most of the variability seen among haplotypes, and the final C-terminal region is most similar to a PAN or apple domain involved in protein-protein or protein-carbohydrate interactions. Of most interest to researchers has been the hyper variable region, where three distinct regions of variability (HVI, HVII and HVIII) have been identified and are thought to be under balancing selection. In addition, a number of specific amino acids are speculated to be under selection to change physiochemical properties, which may explain the evolution of new S haplotypes. Data are presented demonstrating that the majority of SCR binding is focused in the hyper variable subdomain.Researching on self-incompatibility will impact not only on the understanding of SRK and receptor kinase signaling in plants, but will also provide a good tool to enhance forth putting of SI accessions in Brassicas breeding. To identify amino acid fragments within the SRK extracellular domain (eSRK) that are required for ligand-selective activation, we assayed chimeric eSRK between two S-locus haplotype (S7and S28) in Brassica oleracea (inbred lines E and F), and identified the interaction between eSRK chimeras and SCRs by Yeast Two-Hybrid System. To identify residues within the hvⅠ-hvⅡ region that determine SI specificity, we focused on polymorpHic sites that differ between the hvl-hvⅢ regions of eSRKs. The eSRKE were modified by site-directed mutagenesis to generate9mutants containing single-site substitutions at hvⅠ-hvⅢ regions. The interaction between eSRKE mutants and SCRE will be detected by Yeast Two-Hybrid System.1Affinity analysis of plant materialE and F are inbred lines of Brassica oleracea L. var.capitata L. To identify the.compatibility of E,F, we assayed affinity index determination and fluorescence microscopy of pollen germination in situ. Germination assay showed that there were few pollens came from E can germination on E's pistil, and only a few (<10pollen tubes) pollen tubes got through the stigma, as well as F, indicated that E and F wre self-incompatibility lines. when self-fertilization they got a low compatibility index as0.23and0.19, respectively. When cross-pollination, compatibility index of E and F were higher than5, there are more than25pollen tubes could get through the stigma. All those results above suggested that E and F belonged to different S haplotype. 2Cloning of eSRK and SCR genesWe designed specific primers eSRKS/eSRKAS used to clone eSRK cDNA of E, F by RT-PCR base on the relatively conservative regions of eSRK amino acid residue, which named as eSRKE and eSRKF respectively. eSRKE nucleic acid sequence was highly homologous to BoSRK28, and eSRKF nucleic acid sequence was highly homologous to BoSRK7,both of which were determined by BLAST analysis in NCBI. BLAST results suggested that E was S28haplotype, and F was S7haplotype. Two eSRK amino acid sequences containd three subdomains of eSRK proteins, mannose-binding lectins like domain, the middle hyper variable region and PAN-Apple domain, signal peptide of the N-terminal was not included. There were12conserved cysteine residues in eSRK-E and eSRKF amino acid sequences. Primers used to clone SCR gene were based on the nucletides information. SCRE and SCRFwere homologous to BoSP11-28and BoSP11-7, respectively, which were amplified from gDNA of E and F. The SCRF gene encodes53amino acids, the SCRE encoding61amino acids. The two amino acid sequence contained all the amino acids of the mature peptide of SCR. They also contained one glycine residue and eight cysteine residues which were conserved with all other SCR alleles.3Detection of the interaction between chimeric eSRKs and SCRs3.1Detection of the interaction between chimeric eSRKs and SCRs by Yeast Two-Hybrid systemTo study the role of the HV Ⅰ/Ⅱ region in SRK ligand-selective activation, we performed Yeast Two-Hybrid System assay on chimeric eSRK between eSRKE and eSRK-F, and identified the interaction between eSRK chimeras and SCRs. The clones contained pGBKT7-SCRE×pGADT7-eSRKE, pGBKT7-SCRF×pGADT7-eSRKF or positive control pGBKT7-p53×pGADT7-T grown on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plate, and clones contained the others did not grow on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plate. This pHenomenon showed that SRKE (not chimeras) could interact with SCRE, the same as SRKF-SCRF. All of eSRK chimeras could not interact with SCRs. The results demonstrated that HV Ⅰ and HV Ⅱ region were essential for specificity in the SRK-SCR interaction. However, eSRK chimeras could not interact with SCRF, should be due to the overall sequence or3D conformation of the segments which determine SI specificity, although they contained hypervariable regions came from eSRKF.3.2Expression of the chimeric eSRKs and SCRs in E.coli, and detection of the interaction between chimeric eSRKs and SCRs in vitropET43.1(a)+was used as prokaryotic expression plasmid to express His6eSRK fused proteins and chimeric His6eSRKs fused proteins. SCR genes were ligand with pGEX-6p-1expression plasmid, which can express GST fused protein. The E.coli BL21(DE3) contained pET43.1(a)-eSRK (eSRK,eSRKF,eSRKE-1,eSRKE-2and eSRKE-3) plasmid were induced overnight by0.1mM IPTG under25℃. The expreesion products were purified by MagHis M Protein Purification Systerm and analyzed by SDS-PAGE. SDS-PAGE showed that the soluble protein SRKE,eSRKF,eSRKE-1, eSRKE-2and eSRKE-3were expressed at107.7kD. The soluble recombinant SCRE, SCRF were overnight expressed by0.1mM IPTG under16℃. The expression products were purified by MagGSTTM Protein Purification System and analyzed by SDS-PAGE. SDS-PAGE showed that recombinant SCRE and SCRF was expressed at33.9kD and33.0kD receptively.To test the binding specificities of recombinant eSRKs and their chimeras, eSRKE, eSRKF,eSRKE-1,eSRKE-2and eSRKE-3were incubated with an equal amount of SCRE receptively. Bound SCRE was purified by MagGSTTM Protein Purification System and analyzed by SDS-PAGE. SDS-PAGE showed that all of receptors bound SCRE, showed no obvious haplotype specificity. SCRF was able to pull down eSRKE,eSRKF eSRKE-1,eSRKE-2and eSRKE-3, like SCRE. These results indicate that, for these haplotypes at least, eSRKs and their chimeras in isolation retain the ability to bind SCR but do not display S specificity in vitro, although SCRE and SCRF showed unequal efficiency in binding with each receptors.4Detection of the interaction between eSRKE mutants and SCRE by Yeast Two-Hybrid systemTo identify residues determines SI specificity within the hvl-hvⅡ region, we replaced L179,S181,G182,Q184,1248,1252,V253,1264and F269in eSRKE amino acid backbone with Alanine residue receptively. Those mutants were named as M1, M2, M3, M4, M5, M6, M7, M8, M9receptively. To test the ligand-specific activation of eSRKE mutants, each of mutants and SCRE was cotransformed into AH109yeast stain. Transformants were screened on SD/-His/-Trp/-Leu agar plates, then on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plates. All of transformants can grow on SD/-His/-Trp/-Leu agar plates. Except for M1,M3,M5,M6,M9and negative control, all of the other transformants grow on SD/-His/-Trp/-Leu/-Ade/X-a-Gal/25mM3-AT agar plates, and showed different colors from pale blue to dark blue. This result indicated that S181A, Q184A, V253A, I264A mutants showed different efficiency in ligand-specific activation; L179A, G182A, I248A, I252A, F269A mutants did not interact with SCRE.