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. Calcium-dependent protein kinases (CDPKs) decode calcium (Ca) 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 Ca-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.

No abstract

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.