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. 2013 May 21;110(21):8744-9.
doi: 10.1073/pnas.1221294110. Epub 2013 May 6.

Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation

Ullrich Dubiella  1 , Heike Seybold, Guido Durian, Eileen Komander, Roman Lassig, Claus-Peter Witte, Waltraud X Schulze, Tina Romeis
Affiliations

Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation

Ullrich Dubiella et al. Proc Natl Acad Sci U S A. .

Abstract

In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid-mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation _target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant.

Keywords: ROS signaling; disease resistance; plant innate immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CPK5 is rapidly biochemically activated on PAMP elicitation and induces cell death. (A) Flg22 induces biochemical modification of CPK5. On transient expression in Col-0 Arabidopsis mesophyll protoplasts, full-length CPK5 (StrepII tagged) showed biochemical modification, detected 10 min after treatment with buffer (−) or 200 nM flg22 (+) by immunoblot analysis. This effect was not seen with kinase-deficient CPK5m or CPK21. (B) Flg22-induced modification is caused by CDPK phosphorylation. CPK5 prepared as above was affinity purified and incubated for 10 min with λ-phosphatase before immunoblot analysis. (C) PAMP elicitation triggers CPK5 kinase activity. On expression in Col-0 or fls2 protoplasts, CPK5 protein kinase activation was analyzed 10 min after treatment with buffer (−), 200 nM flg22, or elf18 (+) by histone in-gel kinase assay (Upper) and immunoblot analysis (Lower). (D) Constitutively active CPK5-VK induces cell death in Arabidopsis. Transient expression of CPK5-VK, but not kinase-deficient CPK5-VKm, resulted in enhanced cell death development, detected by fluorescence microscopy after staining with fluorescein diacetate and propidium iodide (Upper). Error bars, SD (n = 3); two-way ANOVA, Bonferroni posttest, ***P < 0.001. Protein expression was monitored by immunoblot analysis (Lower).
Fig. 2.
Fig. 2.
CPK5 mediates early defense reactions and pathogen resistance. (A) CPK5, but not kinase-deficient CPK5m, expressed in transgenic Col-0 plants, improves resistance to the bacterial pathogen Pst DC3000. Bacterial counts were taken in 6-wk-old Col-0, 35S::CPK5-YFP#7 and 35S::CPK5m-YFP#15 plants at day 0 and day 3 after inoculation of Pst DC3000. c.f.u., colony forming units. Error bars, SD (n = 12); one-way ANOVA, Dunnett posttest, ***P < 0.001. (B) CPK5 controls expression of the early flg22-induced gene NHL10. Basal gene expression was analyzed by qRT-PCR in 6-wk-old plants of Col-0, cpk5, and rbohd mutants and in transgenic CPK5- and CPK5m-expressing lines. Error bars, SEM (n ≥ 12); ***P < 0.0001. (C) CPK5 triggers flg22-induced ROS production. ROS production was determined over 60 min via a luminol-based assay with and without treatment with 200 nM flg22 in plants as in B and the cross resulting from the rbohd mutant and the transgenic CPK5-expressing line #7. RLU, relative light units; error bars, SEM (n = 8); one-way ANOVA, Dunnett posttest, *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
CPK5 phosphorylates N-terminal serine residues of RBOHD in vitro and in vivo. (A) Protein kinase assay with full-length CPK5 and kinase-deficient CPK5m (affinity purified from Nicotiana benthamiana) in the absence (−) or presence (+) of 10 µM calcium using an N-terminal RBOHD peptide (aa 143–152) as substrate. Error bars, SEM (n =3); ***P < 0,0001. (B–E) Quantification of CPK5-dependent in vivo phosphorylation of RBOHD at S39 (B and D) and S148 (C and E) via SRM mass spectrometry. (B and C) Peptide phosphorylation after transfection of protoplasts with CPK5-VK, kinase-deficient CPK5-VKm, or 10 min after addition of 200 μM H2O2. (D and E) Peptide phosphorylation [normalized ion intensities (nni)] in rosette leaves of 4-wk-old Col-0 and cpk5 plants before and 1 h after treatment with 200 nM flg22 and of transgenic lines 35S::CPK5-YFP#2, 35S::CPK5-YFP#7, and 35S::CPK5m-YFP#15. Error bars, SD (n = 3); one-way ANOVA, Holm-Sidak posttest, **P < 0.01; *P < 0.5.
Fig. 4.
Fig. 4.
CPK5 and RBOHD are required for activation of distal defense responses. (A) Biochemical CPK5 kinase activity is induced by H2O2. Col-0 protoplasts expressing full-length CPK5 were treated for 30 min with buffer (−), 200 μM H2O2, or 200 nM flg22. Expression and CPK5 phosphorylation were monitored by Coomassie staining and immunoblot analysis. CPK5 kinase activity was determined by histone in-gel kinase assay, and fold induction of kinase activity was calculated by phosphoimaging. (B) Scheme of distal response study sampling. (C) Distal flg22-induced NHL10 expression is dependent on CPK5 and RBOHD. Forty-five minutes after 200 nM flg22 injection into a lower rosette leaf of 6-wk-old Col-0, cpk5, and rbohd plants, NHL10 gene expression was quantified by qRT-PCR in an upper leaf. Error bars, SEM (n ≥ 7); Student t test,*P < 0.05. (D) flg22-induced phosphorylation of RBOHD at S39 quantified via directed mass spectrometry in distal leaves harvested 15 min after treatment of lower leaves as under C. Error bars, SD (n = 3); pairwise t tests compared with untreated state, *P < 0.05.
Fig. 5.
Fig. 5.
Model for CPK5 and RBOHD activation circuit facilitating ROS-mediated cell-to-cell signal propagation.
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