Identification and mutational analysis of Arabidopsis FLS2 leucine-rich repeat domain residues that contribute to flagellin perception

Abstract

Mutational, phylogenetic, and structural modeling approaches were combined to develop a general method to study leucine-rich repeat (LRR) domains and were used to identify residues within the Arabidopsis thaliana FLAGELLIN-SENSING2 (FLS2) LRR that contribute to flagellin perception. FLS2 is a transmembrane receptor kinase that binds bacterial flagellin or a flagellin-based flg22 peptide through a presumed physical interaction within the FLS2 extracellular domain. Double-Ala scanning mutagenesis of solvent-exposed beta-strand/beta-turn residues across the FLS2 LRR domain identified LRRs 9 to 15 as contributors to flagellin responsiveness. FLS2 LRR-encoding domains from 15 Arabidopsis ecotypes and 20 diverse Brassicaceae accessions were isolated and sequenced. FLS2 is highly conserved across most Arabidopsis ecotypes, whereas more diversified functional FLS2 homologs were found in many but not all Brassicaceae accessions. flg22 responsiveness was correlated with conserved LRR regions using Conserved Functional Group software to analyze structural models of the LRR for diverse FLS2 proteins. This identified conserved spatial clusters of residues across the beta-strand/beta-turn residues of LRRs 12 to 14, the same area identified by the Ala scan, as well as other conserved sites. Site-directed randomizing mutagenesis of solvent-exposed beta-strand/beta-turn residues across LRRs 9 to 15 identified mutations that disrupt flg22 binding and showed that flagellin perception is dependent on a limited number of tightly constrained residues of LRRs 9 to 15 that make quantitative contributions to the overall phenotypic response.

Figure 1.

Ala-Scanning Mutagenesis Identifies FLS2 LRRs 9 to 15 and Possibly LRR 23 as Regions Required for Plant Response to the flg22 Flagellin Peptide. (A) Positions within the FLS2 LRR domain subjected to double-Ala site-directed mutagenesis. Each mutant allele carried only the two mutations indicated on a single line (i.e., carried Ala codons in place of the codons for the two circled amino acids, altering only one repeat of the LRR per allele). This mutagenesis was not successful for the LRR 10 mutant allele. Numbers at right index the LRR repeats; numbers across the top index residues that received further study. (B) Additional substitutions made for LRRs 5, 6, 23, 24, and 25 targeting L as well as x positions in the LxxLxLxxN consensus motif. Note that letters within circles designate residues encoded by the mutant allele. (C) FLS2-mediated response of Arabidopsis seedlings to flg22 treatment. Mutant fls2-101 plants were transformed with the FLS2 alleles shown in (A) and (B), controlled by the native FLS2 promoter and terminator. Response to flg22 was measured using the seedling growth inhibition assay. Each bar reports the average of 10 or fewer independent T1 plants treated with 10 μM flg22 for 12 d. Error bars indicate se. wt Col-0 transformants received the wild-type Col-0 FLS2 construct. Significant differences from the response conferred by wt Col-0 are marked with asterisks, based on Fisher's protected LSD (P = 0.05). The responses of wild-type Col-0 plants from a separate experiment performed under the same conditions are shown for comparison at far left.

Figure 2.

Brassica FLS2 LRR Domains Confer flg22 Sensitivity in Arabidopsis and Function in Brassica. (A) FLS2 homolog LRR-encoding domains from the indicated B. rapa and B. oleracea sources were cloned in place of the wild-type Arabidopsis Col-0 LRR-encoding domain within Arabidopsis FLS2. FLS2-mediated responses of independent T1 transformants of Arabidopsis fls2-101 expressing the indicated FLS2 transgenes were assayed by measuring seedling growth in the presence or absence of 10 μM flg22 for 12 d. (B) B. rapa and B. oleracea seedlings germinated in 10 μM flg22 and grown for 6 d. Each bar represents 10 to 12 plants. For both (A) and (B), values report means ± se.

Figure 3.

Identification of flg22-Responsive Brassicaceae Accessions. Seeds of single accessions of the indicated species were germinated overnight and then grown in medium with or without 10 μM flg22 for 5 to 12 d depending on the species. Values report means ± se for 12 or fewer plants. Accessions exhibiting significant flg22-induced growth inhibition relative to untreated control are marked with asterisks, based on t tests (P < 0.05).

Figure 4.

Two Methods to Identify Conserved Regions That Are Predicted Functional Sites in the LRR of FLS2. (A) Location of conserved blocks of solvent-exposed β-strand/β-turn LRR residues among FLS2 products from Arabidopsis, Brassica, and other Brassicaceae species. Composite scores for blocks of adjacent residues were calculated by summing individual residue conservation scores from a ClustalX alignment of FLS2 LRR-derived amino acid sequences from flg22-responsive accessions. Entire β-sheet indicates the conservation score for windows of 15 residues: all five solvent-exposed β-sheet residues of that repeat and the two flanking repeats (plotted against the y axis at right). β-sheet left, middle, and right indicate the scores for windows of 9 residues: the first, middle, or last three solvent-exposed β-sheet residues for that LRR and the two neighboring LRRs (plotted against the y axis at left). (B) Location of conserved clusters of spatially proximal FLS2 LRR residues among FLS2 homologs from Arabidopsis, Brassica, and other Brassicaceae species. Three-dimensional models of repeats 2 to 11 and 10 to 19 of the FLS2 LRR were generated based on the structure of bean PGIP. CFG analysis (Innis et al., 2004) was then performed using these models and the ClustalX alignment of FLS2 LRRs from flg22-responsive accessions. Red regions represent highly conserved regions predicted to form protein functional sites, while white is intermediate and blue represents less conserved regions (note: CFG excludes aliphatic residues such as L, I, and V from the analysis). Index numbers are provided at the right of every third LRR repeat. Both (A) and (B) suggest a possible flg22 perception region centered on LRRs 12 to 14.

Figure 5.

Mutational Analysis of Predicted Solvent-Exposed β-Strand/β-Turn Residues of FLS2 LRRs 9 to 15. (A) Functional impact of β-strand/β-turn amino acid substitutions at 34 sites within FLS2 LRRs 9 to 15. Each data point reports the flg22 response of an independent T1 seedling of Arabidopsis fls2-101 transformed with a mutagenized FLS2-HA construct or transformed with wild-type FLS2-HA (WT) or with the empty vector (EV) as controls. Columns of data points correspond to single FLS2 libraries mutagenized at the indicated β-strand/β-turn position (the x axis uses the numbering system of Figure 1A). The y axis reports the FLS2-mediated response as seedling weight after growth in 10 μM flg22, relative to the average growth in flg22 of multiple fls2-101 T1 seedlings transformed with wild-type FLS2-HA and tested on the same date. Controls at far right determined 95% cutoff lines such that relative seedling weight < 1.66 = flg22-responsive and relative seedling weight > 2.82 = flg22-insensitive. Data from one representative experiment are shown. (B) Proportion of mutants at each β-strand/β-turn residue position that showed flg22 insensitivity, reporting data from all experiments performed as in (A). The y axis ratio is the number of T1 seedlings classified as flg22-insensitive divided by the number of T1 seedlings classified as flg22-sensitive. Each bar reports data for a single library that randomized a single β-strand/β-turn solvent-exposed position within FLS2 LRRs 9 to 15. Library index names are shown for the five LRR 12 libraries as an example. Ratios of >1 result when the number of alleles that had lost flg22 responsiveness was greater than the number that retained flg22 responsiveness. WT indicates fls2-101 plants transformed with the wild-type FLS2-HA construct. Sufficient libraries were not available for positions 10.2 and 14.1 (asterisks).

Figure 6.

Mutant FLS2-HA Proteins Were Detectably Expressed in Most flg22-Insensitive Transgenic Seedlings, and Transgenic Seedlings That Expressed Wild-Type FLS2-HA Protein at These Levels Were All flg22-Responsive. (A) Anti-HA protein gel blots of total protein extracts from Arabidopsis fls2-101 seedlings transformed with mutant FLS2 alleles. T1 seedlings that were flg22-insensitive and from a broad set of libraries were otherwise chosen at random for testing. Prior to protein extraction, a small portion of tissue was saved for PCR amplification of the FLS2 transgene and subsequent DNA sequencing. Numbers above the lines indicate the mutant library, and the encoded amino acid change for that allele are listed below the lines. WT indicates the fls2-101 plant transformed with wild-type Col-0 FLS2. Note that for any allele, the level of transgene-derived FLS2-HA will vary between independent transformants, as shown below. (B) Anti-HA protein gel blots of total protein extracts from independent T1 transformants of Arabidopsis fls2-101 transformed with wild-type Col-0 FLS2-HA flanked by native FLS2 promoter and terminator. Seedlings were tested for responsiveness to flg22 prior to protein extraction. All plants that carried a detectable FLS2-HA band were flg22-responsive, while the one plant without a detectable FLS2-HA band was flg22-insensitive.

Figure 7.

Mutations That Disrupt FLS2-Mediated flg22 Binding and Responsiveness. (A) Binding of ¹²⁵I-flg22 in total extracts of Arabidopsis Col-0 or Col-0 fls2-101 transformed with the designated loss-of-function FLS2-HA alleles flanked by native FLS2 promoter and terminator. (B) Protein gel blot of total protein from plant material of (A), probed with anti-HA antibody. Note that Col-0 is wild-type Col-0 rather than a HA-tagged transgenic line. (C) FLS2- and flg22-dependent seedling growth inhibition in transgenic Arabidopsis Col-0 fls2-101 transformed with wild-type or mutant FLS2-HA alleles flanked by native FLS2 promoter and terminator. Seedlings were grown in 10 μM flg22. (D) FLS2-dependent ethylene production after 5 h of exposure to 1.0 μM flg22. Leaf strips were from six separate 5-week-old Arabidopsis plants (described in [C]). (E) Callose deposition visualized as fluorescent spots on leaves of Arabidopsis seedlings (described in [C]) at 24 h after exposure to 3 μM flg22. Two replicate wild-type Col-0 leaves are shown for comparison. (F) Binding of ¹²⁵I-flg22 in total extracts of transgenic Arabidopsis Col-0 fls2-101 transformed with wild-type or mutant FLS2-HA alleles that still respond to flg22. Data were normalized by dividing counts per minute for each data point by the counts per minute for the maximally binding sample (nonradioactive competitor [flg22] = 0.01 nM) for that plant extract to allow comparison of experiments from separate dates.