Structure of the Male Determinant Factor for Brassica Self-incompatibility

Abstract

Many flowering plants possess a self-incompatibility system to prevent inbreeding. In Brassica rapa, self/ non-self recognition in mating is established through S-haplotype-specific interactions between stigma receptors and S-locus protein 11 (SP11, also called S-locus cysteine-rich protein) that is encoded at the highly polymorphic S-locus. Here we describe the solution structure of the SP11 protein of the S 8 -haplotype (S 8 -SP11), which specifically binds to the stigma factor of the same haplotype. It folds into an ␣/␤ sandwich structure that resembles those of plant defensins. Residues important for structural integrity are highly conserved among the allelic SP11s, suggesting the existence of a common folding pattern. Structure-based sequence alignment and homology modeling of allelic SP11 identified a hypervariable (HV) region, which is thought to form a loop that bulges out from the body of the protein that is amenable to solvent exposure. We suggest that the HV region could serve as a specific binding site for the stigma receptor. Self-incompatibility (SI)1 is one of several mechanisms available to flowering plants to prevent inbreeding and thereby promote out-crossing (1). SI is based on self and non-self discrimination processes between pollens and stigmas, followed by selective inhibition of self pollen germination and/or growth. For Brassica rapa, the SI system is controlled sporophytically by multiple haplotypes at a single locus, termed the S-locus. Recognition between pollen and stigma is achieved by gene products of the S-locus, which contains three highly polymorphic genes: SP11/SCR (2, 3), S-locus receptor kinase (SRK) (4), and S-locus glycoprotein (SLG) (5, 6). SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen, whereas SRK encodes a transmembrane receptorlike serine-threonine kinase that determines the S-haplotype specificity of the stigma. SLG is an abundant cell wall-associated glycoprotein similar in sequence to the extracellular domain of SRK.Among these pollen and stigma factors, SRK and SLG, the female factors, exhibited several features typical of SI factors. They are predominantly expressed in stigma papilla cells immediately prior to flower opening concomitant with the acquisition of SI in the stigma. Their sequence variations are correlated with the S-haplotypes. Following the accumulation of such circumstantial evidence, "gain-of-function" experiments employing transgenics established that SRK alone determines the S-haplotype specificity of the stigma, whereas SLG facilitates the recognition process (7). In contrast to these female factors, the identification of the male S determinant remained elusive. Two approaches were used for its eventual identification: one approach concerned the genome analysis of the Slocus region to identify genes specifically expressed in anthers (8), whereas the other employed a fluorescent differential display methodology to identify anther-expressed genes that were specific for the S-haplotype (9). These in...

Structural basis for specific self-incompatibility response in Brassica

“…Tyr52, which is conserved in many Brassica SCRs (Supplementary information, Figure S3A), is sandwiched between the α-helix and the β-sheet (Supplementary information, Figure S3B), and this configuration may also contribute to the structural integrity of SCR9. Database searches revealed that the structure of SCR9 is similar to the structures of SCR8 [22], plant defensins [28], and scorpion neurotoxins [29] (Figure 3C), though the positions of their disulfides bonds are not conserved. Despite this high level of structural similarity, these proteins exhibit strikingly different surfaces (Supplementary information, Figure S3C), which may allow them to have different mechanisms of action and biological activities.…”

“…But whether these regions are generally required for the recognition of all SCRs by their cognate SRKs remains unknown. In the case of SCR variants, which are more polymorphic than SRKs, an NMR study suggested that these small proteins of ~50 amino acids that typically contain eight conserved cysteines may all have a structure similar to defensins [22]. One study of two B. oleracea SCR variants, SCR6 and SCR13, showed that four contiguous amino-acid residues located between the fifth and sixth conserved cysteines are critical for the specific recognition of SCR13 by SRK13 but not for the recognition of SCR6 by SRK6 in planta [23].…”

Diversification and Alteration of Recognition Specificity of the Pollen Ligand SP11/SCR in Self-Incompatibility of Brassica and Raphanus[W]

“…This result suggests that other regions of RsSP11-6 may also be important for the recognition by the BoS-18 stigmas. Mishima et al (2003) have elucidated the solution structure of the SP11 proteins, in which Region III forms an a-helix and Region V corresponds to the region from b2 to b3. Region III and Region V are arranged close to each other by disulfide linkage (Takayama et al, 2001;Mishima et al, 2003).…”

“…Mishima et al (2003) have elucidated the solution structure of the SP11 proteins, in which Region III forms an a-helix and Region V corresponds to the region from b2 to b3. Region III and Region V are arranged close to each other by disulfide linkage (Takayama et al, 2001;Mishima et al, 2003). The lower half of Boxes indicate the conserved Cys residues, and asterisks show the same amino acid residues between two sequences.…”

“…Sato et al (2003) have assigned six regions to SP11, Region I to Region VI, on the basis of conserved Cys residues, and considered Regions III, V, and VI to be more important for recognition specificity than Regions I, II, and IV, because amino acid sequences in Regions III, V, and VI are conserved between the interspecific pairs. Determining the solution structure of the SP11 protein of B. rapa S-8, Mishima et al (2003) have identified a hypervariable region, which they considered to be important for recognition specificity. This hypervariable region corresponds to Region IV, which has been considered to be unimportant by Sato et al (2003).…”