A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana
- University of Florida, United States
- GlaxoSmithKline, United States
Figures
Figure 1
Exogenous NAD+-induced transcriptome changes.
(A) Gene Ontology (GO) term enrichment test of the genes that were upregulated and downregulated by NAD+ treatment at 4 hr showed that genes involved in plant defense such as innate immune response, …
Figure 2 with 2 supplements
LecRK-I.8 functions in extracellular NAD+-triggered defense signaling pathway.
(A) to (C) NAD+-induced expression of PR1 (A), PR2 (B), and PR5 (C) was reduced in the lecrk-I.8 mutants. Plants were treated with 0.2 mM NAD+ solution or water. Leaf tissues were collected 20 hr …
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Figure 2—source data 1
LecRK-I.8 functions in extracellular NAD+-triggered defense signaling pathway.
- https://doi.org/10.7554/eLife.25474.005
Figure 2—figure supplement 1
Exogenous NAD+-induced Psm ES4326 resistance in T-DNA insertion lines of 14 candidate genes and transcript levels of LecRK-I.8 in three T-DNA insertion lines.
(A) Leaves of 4-week-old soil-grown plants were infiltrated with 0.2 mM NAD+ or water. Five h later, the infiltrated leaves were inoculated with a Psm ES4326 suspension (OD600 = 0.001). Eight leaves …
Figure 2—figure supplement 2
Characterization of 35S:LecRK-I.8 transgenic lines.
(A) Expression levels of LecRK-I.8 in 35S:LecRK-I.8-GFP transgenic lines. Total RNA was extracted from the wild type (WT) and 11 single T-DNA insertion homozygous 35S:LecRK-I.8 transgenic lines and …
Figure 3 with 2 supplements
Subcellular localization of the LecRK-I.8-GFP fusion protein.
(A) Putative RGD-binding motifs in DORN1 and LecRK-I.8. (B) Confocal images of N. benthamiana epidermal cells transiently expressing GFP (Left) and LecRK-I.8-GFP (right). (C) Confocal images of N. …
Figure 3—figure supplement 1
Alignment between the NAD receptor LecRK-I.8 and the ATP receptor DORN1.
The amino acid sequences of LecRK-I.8 and DORN1 were aligned using the CLUSTALW tool at the PBIL (Pôle Bioinformatique Lyonnais). LecRK-I.8 and DORN1 (LecRK-I.9) have approximately 81.5% amino acid …
Figure 3—figure supplement 2
Characterization of 35S:LecRK-I.8-GFP lecrk-I.8–2 transgenic lines.
(A) LecRK-I.8-GFP protein levels in 35S:LecRK-I.8-GFP lecrk-I.8–2 transgenic lines. Total protein was extracted from the wild type (WT) and 11 single T-DNA insertion homozygous 35S:LecRK-I.8-GFP …
Figure 4
Kinase activity of the LecRK-I.8 kinase domain.
An autoradiograph (right panel) showing that the kinase domain (KD) of LecRK-I.8 is active based on autophosphorylation and phosphorylation of the myelin basic protein. The purified MBP and …
Figure 5 with 3 supplements
LecRK-I.8 binds NAD+.
(A) to (C), Binding of 32P-labeled NAD+ to immunoprecipitated GFP and eLecRK-I.8-GFP proteins (A), purified eLecRK-I.8-HA-His protein (B), and recombinant MBP, MBP-eLecRK-I.8, MBP-eDORN1, and …
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Figure 5—source data 1
LecRK-I.8 binds NAD+.
- https://doi.org/10.7554/eLife.25474.013
Figure 5—figure supplement 1
Purified eLecRK-I.8 proteins.
(A) Proteins immunoprecipitated from the wild-type (WT), 35S:GFP and 35S:eLecRK-I.8-GFP plants using the anti-GFP antibody. (B) Proteins purified from N. benthamiana leaves infiltrated with …
Figure 5—figure supplement 2
NAD+ binding assay of the recombinant MBP-eLecRK-I.6 protein.
(A) Proteins purified from E. coli cells expressing the recombinant MBP, MBP-eLecRKI.8, and MBP-eLecRK-I.6 fusion proteins using the amylose resin. (B) Binding of 32P-labeled NAD+ to recombinant …
Figure 5—figure supplement 3
Induction of several early PAMP-responsive genes by low concentrations of NAD+.
Four-week-old soil-grown wild-type Col-0 plants were treated with the indicated concentration of NAD+ solution. Leaf tissues were collected 30 min later for qPCR analysis. Expression levels were …
Figure 6
Extracellular NADP+-, flg22-, and SA-induced immune responses are not affected in the lecrk-I.8 mutants.
(A) NAD+-, NADP+-, flg22-, and SA-induced PR1 expression in the wild type (WT) and the lecrk-I.8 mutants. Plants were infiltrated with 0.2 mM NAD+, 0.2 mM NADP+, 1 μM flg22, or water. For SA …
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Figure 6—source data 1
Extracellular NADP+-, flg22-, and SA-induced immune responses are not affected in the lecrk-I.8 mutants.
- https://doi.org/10.7554/eLife.25474.018
Figure 7 with 1 supplement
Immune responses induced by different concentrations of NAD+ in lecrk-I.8–2.
(A) Comparison of different concentrations of NAD+-induced expression of PR1, PR2, and PR5 in lecrk-I.8–2 and the wild type (WT). Leaves of 4-week-old soil-grown plants were infiltrated with the …
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Figure 7—source data 1
Immune responses induced by different concentrations of NAD+ in lecrk-I.8-2.
- https://doi.org/10.7554/eLife.25474.020
Figure 7—figure supplement 1
NAD+-induced expression of several early defense-responsive genes in the lecrk-I.8–2 mutant.
Four-week-old soil-grown wild-type (WT) and lecrk-I.8–2 plants were treated with 0.2 mM NAD+ solution or water. Leaf tissues were collected 4 hr later for qPCR analysis. Expression levels were …
Figure 8 with 1 supplement
Basal immunity is compromised in the lecrk-I.8 mutants.
(A) Psm ES4326-induced PR1 expression was inhibited in the lecrk-I.8 mutants. Plants were inoculated with (+) or without (−) a Psm ES4326 suspension (OD600 = 0.0001). Leaf tissues were collected 24 …
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Figure 8—source data 1
Basal immunity is compromised in the lecrk-I.8 mutants.
- https://doi.org/10.7554/eLife.25474.023
Figure 8—figure supplement 1
Biological induction of SAR in lecrk-I.8 mutants.
Three lower leaves on each plant were inoculated with Psm ES4326 (OD600 = 0.002) (+SAR) or mock-treated with 10 mM MgCl2 (-SAR). Two days later, two upper uninfected/untreated leaves were …
Additional files
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Supplementary file 1
(A) Well-known defense genes affected by NAD+ treatment.
(B) Receptor-like genes induced by NAD+ treatment. (C) Receptor-like kinase genes induced by P. brassicae oviposition and/or NAD+ treatment. (D) Primers used in this study.
- https://doi.org/10.7554/eLife.25474.025
