In vivo detection of residues required for ligand-selective activation of the S-locus receptor in Arabidopsis

Abstract

The self-incompatibility response of crucifers is a barrier to fertilization in which arrest of pollen tube development is mediated by allele-specific interactions between polymorphic receptors and ligands encoded by the S-locus haplotype. Activation of stigma-expressed S-locus receptor kinase (SRK) [1] by pollen coat-localized S-locus cysteine-rich (SCR) ligand [2-5] and the resulting rejection of pollen occurs only if receptor and ligand are encoded by the same S haplotype [4, 6-8]. To identify residues within the SRK extracellular domain (eSRK) that are required for its ligand-selective activation, we assayed chimeric receptors and receptor variants containing substitutions at polymorphic sites in Arabidopsis thaliana[9, 10]. We show that only a small number of the approximately 100 polymorphic residues in eSRK are required for ligand-specific activation of self-incompatibility in vivo. These essential residues occur in two noncontiguous clusters located at equivalent positions in the two variants tested. They also correspond to sites showing elevated levels of substitutions in other SRKs, suggesting that these residues could define self-incompatibility specificity in most SRKs. The results demonstrate that the majority of eSRK residues that show signals of positive selection and previously surmised to function as specificity determinants are not essential for specificity in the SRK-SCR interaction.

Figure 1. The structure and function of chimeric SRK genes

A. The AtS1pr∷eSRKx:AlSRKb genes used for construction of eSRK chimeras. The top diagram shows the structure of a generic AtS1pr∷eSRKx:AlSRKb gene, in which the AtS1 promoter (checkered arrowhead) drives an SRK transcriptional unit with its seven exons and native 3′ untranslated sequences. Exon 1 encodes the AlSRK extracellular domain (eSRK), exon 2 encodes the transmembrane domain (TM), and exons 3-7 encode the kinase domain. The unique SacI restriction site used for construction of chimeras is shown towards the 3′ end of the eSRK. AlSRKb sequences are shown in dark grey and eSRK sequences (from the initiating methionine codon to the unique SacI site) derived from other variants are shown in light grey. Due to the use of the SacI site, all constructs used in this study contain a common 23-amino-acid region derived from AlSRKb (shown in dark grey, spanning the last 23 amino acids of eSRK, i.e. residues 411-434 in SRKb). The middle diagram is a magnified view of the AleSRKb (with numbers indicating amino acids). The vertical lines delineate predicted structural subdomains in the eSRK [29]: SP, signal peptide; LLD1 and LLD2, lectin-like domains 1 and 2; EGF-like, epidermal growth factor-like domain; and PAN_APPLE domain. The locations of hypervariable regions are indicated below the diagrams and correspond to the following amino-acid segments in AlSRKb: 204-219 (hvI), 269-304 (hvII), 326-340 (hvIII). The lower diagrams show the eSRKs of AtS1pr∷AleSRKa:AlSRKb and AtS1pr∷AleSRK16:AlSRKb, two of the constructs used for domain swaps. B. eSRK chimeras. The structures of four functional chimeric eSRKs are shown. The derivation of various segments is shown by different colors or patterns: AleSRKb: grey; AleSRKa: white; AleSRK16: hatched; AleSRK25: stippled; CgeSRK7: slanted bricks. The limits of the swapped region in these and other chimeras analyzed are indicated in Table 1 and their sequences are shown in Figure S2. C. Pollination phenotypes of A. thaliana plants transformed with eSRK chimeras. First- and second-generation transgenic plants expressing each chimera were pollinated using plants expressing the cognate SCR, other SCRs, and wild-type pollen. eSRK chimeras are indicated in the column below the female symbol: a(b)b, AleSRKa(b)b:AlSRKb; 16(b)16, AleSRK16(b)16:AlSRKb; a(7)a, CgeSRKa(7)a:AlSRKb; 16(25)16, AleSRK16(25)16:AlSRKb. The SCR variants expressed in pollen used for pollination assays are indicated in the row to the right of the male symbol and correspond to the constructs shown in Figure S1: a, native AlSCRa; 25, native AlSCR25; 7, AlSCRb:CgSCR7; 16, AlSCRb:AlSCR16; b, native AlSCRb. The numbers in parentheses show the number of T1 plants that expressed an incompatibility response towards pollen expressing cognate SCR over the total number of primary transformants analyzed. 0 = an incompatible response (typically <5 pollen tubes per pollinated stigma); +++ = a compatible response (typically >50 pollen tubes per pollinated stigma). For each construct, although the majority of transformants exhibiting SI expressed a strong SI response (<5 pollen tubes per pollinated stigma), typically 1 or 2 transformants exhibited a weaker SI response (5-10 pollen tubes per pollinated stigma).

Figure 2. Structure and immunoblot analysis of eSRKa(7)a:SRKb and eSRK16(25)16:SRKb substitution mutants

A. Single-codon substitutions in eSRK chimeras. The diagrams show the specificity-determining hvI-hvIII regions of the eSRKa(7)a and eSRK16(25)16 chimeras, with amino-acid residues depicted by vertical bars. The bottom diagram shows the location of the LLD2 and EGF-like domains and hypervariable regions hvI, hvII, and hvIII. Residues that do not differ between eSRKa and eSRKa(7)a or between eSRK16 and eSRK16(25)16 are shown by light grey bars. Polymorphic residues that were modified by substitution mutagenesis are shown by dark grey and black bars. Each of these variable residues, with the exception of 2 residues in eSRK16(25)16 (marked by gray circles, mutants of which failed to generate transgenic plants), were individually replaced in eSRKa(7)a with residues found at the equivalent positions in eSRKa, and in eSRK16(25)16 with residues found at the equivalent positions in eSRK16. Transgenic stigmas expressing each of the single-codon substitution eSRKa(7)a or eSRK16(25)16 derivatives were tested by pollination with SCR7- or SCR15-expressing pollen, respectively. For most substitution mutants (substituted residues shown as dark grey bars), the stigmas of at least some transformants [13-88% of transformants for eSRKa(7)a mutants and 12-71% of transformants for eSRK16(25)16 mutants] exhibited an incompatible response. For each of the substitution mutants that failed to confer an incompatibility response, between 10 and 18 independent transformants were assayed. Amino-acid residues found to be required for the function of eSRK chimeras are shown by black bars with arrows indicating the amino-acid substitution that caused loss of chimera function. The L218V substitution in eSRKa(7)a and the Q285E substitution in eSRK16(25)16 conferred a weakened incompatibility response (Figure S3). The X298A change in eSRKa(7)a involved inserting alanine at amino-acid site 298. Note that substitutions at two sites, 213 and 301, disrupted the function of both eSRKa(7) and eSRK16(25)16 chimeras: site 213 was sensitive to a change from the polar and charged lysine to a non-polar and uncharged methionine or phenylalanine, while site 301 was sensitive to changes in volume. B. Immunoblot analysis of eSRK chimeras. For immunoblot analysis of HA-tagged eSRKa(7)a chimeras, proteins were extracted from the stigmas of plants transformed with AtS1pr∷eSRKa(7)a:AlSRKb (wt) and its single- and multiple-codon substitution derivatives, and subjected to protein immunoblot analysis (Supplemental Data). The “wt” lane shows the level of non-mutated “wild type” eSRKa(7)a protein found in stigmas exhibiting an incompatibility response toward SCR7-expressing pollen. The remaining lanes show representative patterns for eSRKa(7)a substitution derivatives: nine single-codon substitution derivatives labeled according to the amino-acid substitution introduced into each chimera (numbering as in panel A), and a multiple-codon substitution derivative (multiple). The dashed box indicates the substitution derivatives that did not confer an incompatible response towards SCR7-expressing pollen. The blot was probed sequentially with an anti-HA monoclonal antibody (top panel) and an anti-actin antibody as loading control (bottom panel). The arrow indicates the full-length eSRKa(7)a:SRKb receptor and the asterisk indicates the alternative smaller SRK products typically produced in stigmas [39].

Figure 3. Extent of variability at individual sites within the hvI-hvIII region observed in pairwise alignments of closely-related eSRK pairs

eSRK sequences from Arabidopsis lyrata, A. halleri, Capsella grandiflora, Brassica oleracea, B. rapa, and Raphanus sativus were analyzed by pairwise alignment of the most closely-related sequences that are either known or likely to encode different SI specificities (Figure S4). Pairwise consensus sequences were generated and aligned (Figure S4), and the percentage of consensus sequences that differed at a particular site was calculated. Each site was assigned a “substitution score” between 0 and 100, as shown on the y-axis: a score of “0” indicates that 0% of the variant pairs differ at that site, and a score of “100” indicates that 100% of the pairs differ at that site. The x-axis indicates amino-acid site number along the hvI-hvIII region after removal of indels (Figure S4); this numbering was used to highlight the overlap and clustering of highly variable residues relative to the essential residues identified in planta. The short dark bars indicate the number of sequence pairs with substitutions for each site. Asterisks and circles indicate the residues found to be essential for the function of the eSRK16(25)16 and eSRKa(7)a chimeras, respectively. The locations of the essential sites using eSRKa as a reference sequence (Figure 2) are: K213, I217, L218, P294, X298, D300, and Y301 in eSRK7, and K213, Q285, M289, S292, H295, and V301 in eSRK25. Note that the hvI and hvII regions, and in particular clusters of sites in the vicinity of the essential residues identified in planta, are enriched for residues showing elevated variability relative to other segments, as indicated by the number of residues that are polymorphic in more than 50% of the pairwise alignments (indicated by the horizontal dashed line). Substitution scores for the essential sites identified in vivo are significantly different from the non-essential sites (see Figure S4).