Abstract
Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants’ signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.
Highlights
Tyrosine (Tyr) phosphorylation (TP) is a notable regulator of signal transduction in eukaryotic cells (Blume-Jensen and Hunter, 2001)
To investigate the roles of CRK-promoted TP in ethylene response factor 13 (ERF13) transactivation, CRKs were expressed together with ERF13 as an activator of a reporter gene coexpressed under the control of a chimeric promoter that consisted of four inverted repeats of GCC-box [ERF-binding cis-element (Fujimoto et al, 2000)] fused to a minimal TATA-box, in Arabidopsis mesophyll protoplasts (Figure 1A)
Tyr phosphorylation mediated by Arabidopsis CRKs appeared to modulate the activities of transcription factors (TFs) including ERF13, WRKY DNA-binding protein 14 (WRKY14), and RAP2.6 (Figures 1, 2)
Summary
Tyrosine (Tyr) phosphorylation (TP) is a notable regulator of signal transduction in eukaryotic cells (Blume-Jensen and Hunter, 2001). Nemoto et al (2015) recently reported that Arabidopsis calcium-dependent protein kinase (CPK)-related protein kinases [CRK2 (At3g19100) and CRK3 (At2g46700)] phosphorylate Tyr residues of beta-tubulin and an array of transcription factors (TFs), including ethylene response factor (ERF13) (At2g44840), WRKY DNA-binding protein (WRKY14) (At1g30650), ERF subfamily B-4 member ERF/AP2 transcription factor 2.6 (RAP2.6) (At1g43160), and cryptochrome-interacting basic-helix-loop-helix 5 (CIB5) (At1g26260). In spite of the structural similarity of CRKs to typical Ser/Thr-type protein kinases, CRK2 and CRK3 preferentially phosphorylate tyrosine residues in the absence of calcium (Nemoto et al, 2015)
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