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. 2022 Sep 22;11(19):2960. doi: 10.3390/cells11192960

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Two conserved phenylalanine residues in the malectin domain of CORK1 are important for COM perception. (A) Alignment of the amino acid sequences from malectin of X. laevis and the malectin domains present in A. thaliana LRR-malectin RLKs. Shown here are amino acids from position 101–150 of the alignment. Black shade indicates conserved amino acid residues over 90% threshold. The two conserved phenylalanine residues are indicated with an asterisk. (B) Cytoplasmic calcium elevation by 10 µM CT in root tissue of EMS71 complemented with CORK1, or with single (CORK1^F520A/CORK1^F539A) or double (CORK1^F520AF539A) mutation in the two conserved phenylalanine residues. Error bars represent SE from 8 seedlings. Arrow indicates the onset of elicitor application. Statistical significance at the peak value was determined by Tukey’s HSD test with p ≤ 0.05 and is indicated by different lowercase letters. The experiment was repeated 3 times with similar results. (C) GFP signal in the roots of the wild-type Col-0 (WT) and the transformed EMS71 mutants. EV: Empty vector; EMS71 transformed with the construct 35S::CORK1 with two stop codons after the coding region. The plasma membrane was stained with RH414, and the overlapping signals from GFP and RH414 at the plasma membrane are indicated with white arrows. (D–G) Protoplasts from A. thaliana Col-0 were transfected with the (D) pFRK1::Luciferase (pFRK1::LUC) reporter construct, (E) the reporter construct plus the CORK1 receptor or (F) the reporter construct plus the double-mutated version CORK1^F520AF539A. Results show luciferin-dependent light emission over time after treatment with water (Mock) or 1 µM CT. Arrow indicates the onset of elicitor application at 0 h. Each datapoint represents the mean value from 4 technical replicates. Error bars represent SE. Statistical significance was determined by Student’s T-test between the two treatments (* p ≤ 0.05; ** p ≤ 0.01). The experiment was repeated 4 times with similar results. (G) Western blot indicating the expression of both versions of GFP-tagged CORK1; ctrl: control, protoplast transfected with pFRK1::LUC reporter construct only.

Two conserved phenylalanine residues in the malectin domain of CORK1 are important for COM perception. (A) Alignment of the amino acid sequences from malectin of X. laevis and the malectin domains present in A. thaliana LRR-malectin RLKs. Shown here are amino acids from position 101–150 of the alignment. Black shade indicates conserved amino acid residues over 90% threshold. The two conserved phenylalanine residues are indicated with an asterisk. (B) Cytoplasmic calcium elevation by 10 µM CT in root tissue of EMS71 complemented with CORK1, or with single (CORK1^F520A/CORK1^F539A) or double (CORK1^F520AF539A) mutation in the two conserved phenylalanine residues. Error bars represent SE from 8 seedlings. Arrow indicates the onset of elicitor application. Statistical significance at the peak value was determined by Tukey’s HSD test with p ≤ 0.05 and is indicated by different lowercase letters. The experiment was repeated 3 times with similar results. (C) GFP signal in the roots of the wild-type Col-0 (WT) and the transformed EMS71 mutants. EV: Empty vector; EMS71 transformed with the construct 35S::CORK1 with two stop codons after the coding region. The plasma membrane was stained with RH414, and the overlapping signals from GFP and RH414 at the plasma membrane are indicated with white arrows. (D–G) Protoplasts from A. thaliana Col-0 were transfected with the (D) pFRK1::Luciferase (pFRK1::LUC) reporter construct, (E) the reporter construct plus the CORK1 receptor or (F) the reporter construct plus the double-mutated version CORK1^F520AF539A. Results show luciferin-dependent light emission over time after treatment with water (Mock) or 1 µM CT. Arrow indicates the onset of elicitor application at 0 h. Each datapoint represents the mean value from 4 technical replicates. Error bars represent SE. Statistical significance was determined by Student’s T-test between the two treatments (* p ≤ 0.05; ** p ≤ 0.01). The experiment was repeated 4 times with similar results. (G) Western blot indicating the expression of both versions of GFP-tagged CORK1; ctrl: control, protoplast transfected with pFRK1::LUC reporter construct only.