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. 2023 Jun 2;23(1):294.
doi: 10.1186/s12870-023-04300-0.

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype

Liming Zhao  1   2   3 , Hao-Jie Wang  4   5 , Patricia Dalcin Martins  5 , Joost T van Dongen  5 , Anthony M Bolger  5   6 , Romy R Schmidt  5   7   8 , Hai-Chun Jing  9   10 , Bernd Mueller-Roeber  1   2   11 , Jos H M Schippers  12
Affiliations

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype

Liming Zhao et al. BMC Plant Biol. .

Abstract

Background: Plant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens.

Results: Here, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000.

Conclusion: We demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Keywords: Arabidopsis; Autoimmunity; Onset of leaf death; Receptor-like kinase; Salicylic acid.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The early cell death phenotype of old12. A Image of 4-week-old Arabidopsis thaliana wild-type (WT) and old12 plants grown in long-day photoperiod (16 h). Scale bar = 1 cm. B Number of yellow (/senescent) leaves at different time points after the sowing of wild-type plants and old12. Each data point represents the means of three replicates of ten plants each ± SD. Different letters indicate significant difference according to one-way ANOVA and post-hoc Tukey HSD test. C Progression of senescence and cell death in the second leaf pair of old12 and wild type from 19 to 35 DAS (days after sowing). Scale bar = 1 cm. D, E Chlorophyll (Chl) content and ion leakage in the second leaf pair of old12 and wild type (WT) from 19 to 39 DAS. Each data point represents the mean ± SD from three replicates of six leaves each. Asterisk indicates significant difference (p ≤ 0.05; Student´s t-test) between WT and old12 at the indicated time points. F Cell death staining (trypan blue) of the third leaf of wild-type and old12 leaves at 23 DAS. Arrows indicate lesions. Scale bar = 5 mm
Fig. 2
Fig. 2
The old12 mutant displays a low-temperature-induced lesion mimic phenotype. A Panel I, 28 DAS old12 and wild-type (WT) plants grown in long-day photoperiod (16 h) at 28 °C. Panel II, Phenotype of old12 and WT plants grown for 28 days + 14 days at 28 °C. Panel III, Transfer of old12 plants grown for 28 days at 28 °C to 16 °C for 14 days results in stunted growth and an early onset of senescence as compared to WT. Scale bar = 1 cm. B Chlorophyll (Chl) content in the fifth and sixth leaf of old12 and WT at the different growth conditions shown in (A). Box plots show median and interquartile ranges (IQR), outliers (> 1.5 times IQR) are shown as circles. C Expression of SA pathway and defense-related genes in old12 and WT leaves grown for 28 days at 28 °C (I), or 28 days at 28 °C plus 2 days at 28 °C (II), or 28 days at 28 °C plus 2 days at 16 °C (III). The relative expression level is shown as mean ± SD of three biological replicates. Expression level for each time point was normalized against ACTIN2 (ACT2). Asterisks indicate significant difference under the conditions tested between old12 and wild type for the genes indicated (Student’s t-test; p ≤ 0.05)
Fig. 3
Fig. 3
Identification of the old12 mutation by whole-genome sequencing. A Genome-wide SNP frequency plot of the Arabidopsis genome using a bin size of 1 Mb for the old12 x Col-0 mapping population. Two candidate regions were identified for the old12 locus, one on chromosome I and one on chromosome III. B Genomic representation of the point mutation identified in P2K2 (AT3G45430). Light blue indicates exon sequence and gray corresponds to untranslated regions. C 32 DAS wild type (WT), old12 and two independent complementation (old12 gOLD12) lines grown under long-day photoperiod (16 h). Complementation of old12 with the genomic region containing AT3G45430 restores the wild-type phenotype. Scale bar = 1 cm. D Chlorophyll content (SPAD units) of the 3rd/4th leaf from WT, old12 and old12 gOLD12 #1. Each data point represents the mean ± SD from 10 leaves. E Ion leakage of the 3rd/4th leaf from WT, old12 and old12 gOLD12 #1. Each data point represents mean ± SD from 6 replicates of three leaves each. Asterisks indicate significant difference from WT (Student’s t-test; p ≤ 0.05)
Fig. 4
Fig. 4
The old12 mutation impairs P2K2 kinase activity. A Schematic representation of the P2K2 protein structure highlighting the different domains and the position of the old12 mutation (C407Y, asterisk). B Multiple sequence alignment of the active site of several LecRK proteins and other receptor like kinases (RLKs). The old12 mutation affects a cysteine residue that is conserved in the kinase domain. C P2K2 autophosphorylation activity was analyzed using an expressed and purified FLAG-tagged kinase domain (KD) of the wild-type P2K2 and the old12 mutant. Autophosphorylation was measured by detecting thiophosphorylation with ATPγS and subsequent western blotting using thiophosphate ester-specific antibodies. D Transphosphorylation assays were performed by incubating purified FLAG-tagged KDs of wild-type P2K2 and old12 with the universal kinase substrate MBP. Thiophosphorylation of MBP was subsequently detected by a western blot using antibodies against the thiophosphate ester. The upper arrow indicates autophosphorylation of P2K2, while the lower arrow indicates the transphosphorylation of MBP. Mock reactions only contained the MBP substrate. The loading control involves the detection of OLD12 proteins with an anti-FLAG antibody. Images for the loading controls are cropped, full images can be found in the supplement (Fig. S6)
Fig. 5
Fig. 5
Tissue-specific expression pattern of P2K2. A The expression pattern of P2K2 was analyzed using a GUS reporter driven by a 2.1-kb promoter of P2K2. Two-week-old plants were analyzed. Arrowheads indicate detection of expression in root tips, root epidermis layer and leaf. B As compared to P2K1, P2K2 is weaker expressed, suggesting that P2K1 might be the main extracellular ATP sensing kinase. The image was created with the eFP2 browser (http://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html). C Subcellular localization of P2K2-YFP in Arabidopsis mesophyll protoplasts. YFP fluorescence was observed at the plasma membrane. Chl: chlorophyll autofluorescence; BF: bright field. Scale bar = 15 µm
Fig. 6
Fig. 6
The old12 auto-immunity phenotype relies on SA. A Callose depositions as visualized by aniline blue staining (light blue fluorescence) of 3rd and 4th leaves of 24 DAS (long-day) plants. Scale bar = 200 µm. B PR gene expression in 27 DAS plants grown on soil under long-day condition. Expression data represents means ± SD from three biological replicates. Expression level for each gene was normalized against ACT2. Asterisks indicate significant difference from WT (Student’s t-test; p ≤ 0.05). C Expression of SA biosynthesis genes in Ler-0 and old12 mutants. Bars represent mean ± SD from three biological replicates. Asterisks indicate significant difference from WT (Student’s t-test; p ≤ 0.05). D Crossing nahG into the old12 mutant background reverts the early-senescence phenotype. An F2 population of nahG x old12 was phenotyped and genotyped to determine the presence of old12 SNPs, and the presence of the nahG gene. In the table, a ´ + ´ indicates the presence of either the wild-type and/or old12 allele, and/or the nahG gene. Early senescence is indicated by a yellow score in the table, a green score indicates no early senescence, whereby a dark green color highlights plants that are homozygous for the old12 mutation and at the same time contain the nahG transgene. Shown are plants grown under long-day conditions at 28 DAS
Fig. 7
Fig. 7
old12 plants show increased resistance against Pst DC3000. A Seedlings were inoculated with water (Mock) or 5 × 10.5 bacteria/ml of Pst DC3000 for 4 days. Experiments were repeated twice and showed similar results. B The colony-forming units (cfu) of Pst DC3000 in old12 are significantly different from WT after one day and four days post infection. Each column represents the mean ± SE from 6 biological replicates; the experiment was repeated twice and revealed similar results. Asterisks indicate significant difference between old12 and wild-type plants (Student’s t-test; p ≤ 0.05). DPI: days-post-inoculation C Ion leakage of Ler-0 and old12 seedlings after 4 days of incubation with Pst DC3000. Data represent means ± SE from 6 biological replicates. (* p ≤ 0.05; Student's t-test)
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