Gene cloning, protein expression and purification

The CpWAK genomic sequences were obtained by PCR from genomic DNA isolated from C. plantagineum leaves. The primers used are shown in Table S1. The PCR products were cloned into pJET 1.2 vectors using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific, St LeonRot, Germany) and the PCR fragments were sequenced.

The cDNA fragments encoding CpWAK1EX (amino acids 31–315), CpWAK2EX (amino acids 37–333), R-1 (amino acids 31–160), R-2 (amino acids 161–315) and R-3 (amino acids 31–220) (Fig. 3a) were amplified from a CpWAK1 cDNA clone (Giarola et al. 2016) using primers to add an XhoI site at the 3′ end (CpWAK1_XhoI_R, CpWAK2_XhoI_R, R-1, R-2-rev, R-3, Table S1). An NcoI site is already present in the sequences of CpWAK1, CpWK2, R-1 and R-3. An NcoI site was added at the 5′ end of the R-2 sequence with the primer, R-2-for (Table S1). The NcoI/XhoI fragments were cloned into the expression vector pET28a( +) (Novagen, Darmstadt, Germany) and transformed into BL21 (DE3) E. coli cells (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The fusion constructs, pET28_CpGRP1His and pET28 CpGRP1_N-terminal His, were provided by Giarola et al. (2016) and Jung et al. (2019), respectively. Over-expression and purification of the fusion proteins were performed as described by Jung et al. (2019).

CpWAKs show higher affinity to the pectins in “egg-box” conformation. a Domain structures of CpWAK proteins and the fragments for His-tagged recombinant proteins. The extracellular domains of CpWAK proteins without signal peptides are CpWAK1EX (amino acid 31–315) and CpWAK2EX (37–333 for CpWAK2EX). R-1, R-2 and R-3 are truncated fragments of the CpWAK1EX protein, containing the amino acids 31–160, 161–315 and 31–220, respectively. b The CpWAK1 extracellular domain only binds to the pectin extracts of C. plantagineum leaves. c Both saponification and Ca²⁺ are necessary for higher binding capacity of the CpWAK1 extracellular domain to polygalacturonic acid and commercial pectin. S ± : saponificated/non-saponificated pectins; Ca²⁺  ± : buffer with or without Ca²⁺. d Different subdomains of CpWAK1 showed different pectin binding capacity. WAK1EX, WAK2EX, R-1, R-2 and R-3 represent different fragments as shown in a. Polygalacturonic acid (PGA, Sigma), commercial pectin (pectin from citrus peel, Sigma) and C. plantagineum pectin isolated from C. plantagineum leaves with CDTA (1, 2-cyclohexanediaminetetraacetic acid) solution were normalized by pectin estimation assay, and immobilized in ELISA plate wells, incubated with 0.2 µg of purified recombinant CpWAK1EX or the same amount of other recombinant proteins. The bound recombinant proteins were detected with His-tag antibody (1:10,000). All the mean absorbance values were calculated from three biological of which each included three technical repetitions. Error bars indicate SEM and Mock indicates only buffer without pectin (ns means no significant, ***P < 0.0001, **P < 0.01, t test compared to Mock in b and S-/Ca²⁺- in c, respectively). The letters in d show the significance determined by one-way ANOVA with Bonferroni’s post-test (a–d P < 0.01)