Abstract
Key message
We describe the potato CDPK family and place StCDPK7 as a player in potato response to Phytophthora infestans infection, identifying phenylalanine ammonia lyase as its specific phosphorylation target in vitro.
Abstract
Calcium-dependent protein kinases (CDPKs) decode calcium (Ca2+) signals and activate different signaling pathways involved in hormone signaling, plant growth, development, and both abiotic and biotic stress responses. In this study, we describe the potato CDPK/CRK multigene family; bioinformatic analysis allowed us to identify 20 new CDPK isoforms, three CDPK-related kinases (CRKs), and a CDPK-like kinase. Phylogenetic analysis indicated that 26 StCDPKs can be classified into four groups, whose members are predicted to undergo different acylation patterns and exhibited diverse expression levels in different tissues and in response to various stimuli. With the aim of characterizing those members that are particularly involved in plant–pathogen interaction, we focused on StCDPK7. Tissue expression profile revealed that StCDPK7 transcript levels are high in swollen stolons, roots, and mini tubers. Moreover, its expression is induced upon Phytophthora infestans infection in systemic leaves. Transient expression assays showed that StCDPK7 displays a cytosolic/nuclear localization in spite of having a predicted chloroplast transit peptide. The recombinant protein, StCDPK7:6xHis, is an active Ca2+-dependent protein kinase that can phosphorylate phenylalanine ammonia lyase, an enzyme involved in plant defense response. The analysis of the potato CDPK family provides the first step towards the identification of CDPK isoforms involved in biotic stress. StCDPK7 emerges as a relevant player that could be manipulated to deploy disease resistance in potato crops.
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Acknowledgements
RMU and MES are members of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); RMU is Associate Professor at Universidad de Buenos Aires (UBA). EF, FS, and FGM are fellows from CONICET. We would like to thank Dr. Angelika I. Reichert, Dr. Jun-Jie Zou, Dr. Christian Jelich-Ottmann, and Dr. Diego Wengier for providing the vectors: pET-15b-NtPAL1-4, pGEX-4t-AtHSP1, pGEX-4t-NpPMA2, and pENTR/D-TOPO-YFP, respectively. We also thank Dr. Adriana Andreu for sharing P. infestans isolate and Salome Prat for lending us the cDNA libraries. This work was funded by Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad de Buenos Aires, and Agencia Nacional de Promoción Científica y Tecnológica.
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Communicated by Howard S. Judelson.
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Fig. S1 Recombinant StCDPK7:6xHis production and purification. a StCDPK7:6xHis (MW = 66 kDa) protein purification steps are shown in the Coomassie Brilliant Blue stained gel and in the western blot revealed with a commercial anti-His antibody. NI, non-induced culture; I, IPTG-induced culture; M, membrane extract; S, soluble extract; FT, flow-through; W1-W3, washes; E, eluted fraction. b StCDPK7:6xHis Ca2+-dependence. StCDPK7:6xHis Kinase Activity (µmol min−1 mg−1) was determined in the presence of increasing Ca2+ concentrations using Syntide-2 and 50 µM ATPγP32 as acceptor and donor substrates, respectively. (TIFF 2505 kb)
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Fig. S2 Expression analysis of StCDPKs. a PCRs were performed with specific primers that amplify each newly identified CDPK (6 to 26) using DNA from S. phureja as template. b Semiquantitative RT-PCR assays were performed, with the same specific primers as in a, using cDNA libraries from tuberizing stolons or leaves as template. Specific primers (R5/R4), which amplify StCDPK1/2/3 were used as positive controls (C +). (TIFF 3257 kb)
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Fig. S3 Alignments of StCDPK7, AtCPK1 and NtCDPK2, N-terminal variable domains. The glycine (G) in position 2 and the cysteine (C) in position 4 are highlighted in bold. Conserved autophosphorylation sites, serine (S) or threonine (T) residues, are shaded in black. The arrow indicates the position of S-40 in NtCDPK2 and the corresponding residues in the other CDPKs. PEST motifs are shaded in gray. Protein kinase subdomains I (GQGQFG) are highlighted in bold (TIFF 2402 kb)
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Table S1 Accession numbers of the CDPKs DNA coding sequences from Arabidopsis, tobacco, tomato, and rice, used to perform the phylogenetic tree a (https://www.ncbi.nlm.nih.gov/nucleotide/) (TIFF 3494 kb)
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Table S2 List of primers used in this article. Listed are the primers used for expression analysis of the different CDPKs, GAPDH, EF, PAL, and PR-1b genes, and those used for cloning StCDPK7. Restriction sites are shaded in gray and cloning vectors are indicated. aAccession numbers of the sequences (https://www.ncbi.nlm.nih.gov/nucleotide/) are included. (TIFF 5913 kb)
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Table S3 Myristoylation and Palmitoylation predictions for the CDPK/CRK potato family members. The N-terminal sequences (first 18 amino acids) of the corresponding proteins are shown; the G or C residues in bold indicate putative myristoylation or palmitoylation sites; highlighted in gray are the N residues which, according to Yamauchi et al. (2010), negatively affect myristoylation. The myristoylation sites were predicted by aNMT-The MYR Predictor (http://mendel.imp.ac.at/myristate/cgi-bin/myr_pred.cgi), or by the bMyristoylator program (http://web.expasy.org/cgi-bin/myristoylator/myristoylator.pl). Myristoylation sites predicted as ‘RELIABLE’ comply with the sequence motif as implemented in the present version of NMT-The MYR predictor. Myristoylation sites predicted in the ‘TWILIGHT ZONE’ have a less complete concordance with the myristoylation sequence pattern as implemented in the predictor. In Myristoylator, the score (S) is based on the average responses of 25 artificial neural networks. S = Positive – Negative. For positive scores: 0.0 < S < 0.4 — > Low Confidence; 0.4 < S < 0.85 – > Medium Confidence; 0.85 < S < 1 — > High Confidence. Negative scores, non-myristoylated. cThe palmitoylation sites were predicted by CSS-Palm 3.0 (http://csspalm.biocuckoo.org/) (TIFF 30001 kb)
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Table S4 Prediction of PEST motifs in the CDPK/CRK potato family. PEST motifs were predicted by (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind), as potential proteolytic cleavage sites. Potential PEST motifs together with their PEST score, mass percent of DEPST, and their hydrophobicity index are shown (TIFF 2339 kb)
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Table S5 Cis-ELEMENTS predicted in StCDPK6/7/17/18/21/22 and 25 promoters that could be involved in biotic stress responses. Elements also present in PALs promoters are indicated in bold letters. The number of sites of StCDPK7 promoter is shaded in gray. aData bases: Plant CARE, (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE Website, (https://www.hsls.pitt.edu/obrc/index.php/). Motif occurrences with p values < 0.0001 were chosen and computed using FIMO tool from MEME Suite (http://meme-suite.org/doc/fimo.html?man_type=web) (TIFF 4081 kb)
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Table S6 Cis-ELEMENTS predicted in StCDPK7 promoter. aData bases: Plant CARE, (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE Website, (https://www.hsls.pitt.edu/obrc/index.php/). Motif occurrences with p values < 0.0001 were chosen and computed using FIMO tool from MEME Suite (http://meme-suite.org/doc/fimo.html?man_type=web) (TIFF 3285 kb)
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Fantino, E., Segretin, M.E., Santin, F. et al. Analysis of the potato calcium-dependent protein kinase family and characterization of StCDPK7, a member induced upon infection with Phytophthora infestans . Plant Cell Rep 36, 1137–1157 (2017). https://doi.org/10.1007/s00299-017-2144-x
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DOI: https://doi.org/10.1007/s00299-017-2144-x