Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defense response against Pseudomonas syringae Gennady V. Pogorelko a,b, ⁎, Maria Mokryakova b , Oksana V. Fursova c , Inna Abdeeva b , Eleonora S. Piruzian b , Sergey A. Bruskin b a 219 Bessey Hall, Department of Plant Pathology and Microbiology, Iowa State University, Ames 50014, IA, USA b NI Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow 119991, Russia c Geocryology Department, Moscow State University, Leninskie Gory 1, Moscow 119992, Russia abstract article info Article history: Accepted 10 January 2014 Available online xxxx Keywords: Immunophilin family Plant–pathogen interaction Plant immune system Plant immunophilins are a broadly conserved family of proteins, which carry out a variety of cellular functions. In this study, we investigated three immunophilin genes involved in the Arabidopsis thaliana response to Pseudomo- nas syringae infection: a cytoplasmic localized AtCYP19, a cytoplasmic and nuclear localized AtCYP57, and one nucleus directed FKBP known as AtFKBP65. Arabidopsis knock-out mutations in these immunophilins result in an increased susceptibility to P. syringae, whereas overexpression of these genes alters the transcription profile of pathogen-related defense genes and led to enhanced resistance. Histochemical analysis revealed local gene ex- pression of AtCYP19, AtCYP57, and AtFKBP65 in response to pathogen infection. AtCYP19 was shown to be involved in reactive oxygen species production, and both AtCYP57 and AtFKBP65 provided callose accumulation in plant cell wall. Identification of the involvement of these genes in biotic stress response brings a new set of data that will advance plant immune system research and can be widely used for further investigation in this area. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Biotic stresses caused by phytopathogens can have strong impacts on the growth and development of plants, including crop species. In order to survive, plants have evolved a sophisticated defense system against a variety of pathogens, including viruses, bacteria, fungi, and nematodes (Hou et al., 2009). Stress is recognized and transmitted by the signal transduction system which influences the regulatory ele- ments of stress-inducible genes involved in retrograde signaling to the specific genes and proteins that provide stress resistance (Chen et al., 2002; Knight and Knight, 2001). Studying the functions of stress- inducible genes enables an understanding of the underlying mecha- nisms of plant–stress interactions and modulation of their function by molecular genetic approaches (Sekhar et al., 2010). Efficient detection of pathogens and rapid activation of the plant im- mune system are extremely important for the survival of plants. Patho- gens can be recognized by the perception of conserved microbial molecules named pathogen-associated molecular patterns (PAMPs). Specific PAMPs are detected via corresponding trans-membrane pattern recognition receptors (PRRs) and initiate intracellular immune responses (Zipfel, 2008). These responses include the generation of reactive oxygen species (ROS), protein phosphorylation (Gimenez- Ibanez and Rathjen, 2010), and downstream activation of signaling cascades. Mitogen-activated protein kinase (MAPK) signaling plays a central role in plant response against pathogen intrusion. This pathway starts with initiation by a PRR responsive gene called MEKK which provides a downstream MAP kinase signaling that leads to specific patterns of stress-responsive gene expression as well as changes in post-translational modifications. The immunophilin protein family functions as receptors for immunosuppressive drugs, and has been found in a broad range of organisms, including bacteria, fungi, animals, and plants. Two groups of immunophilin receptors exist in plants: cyclosporin A receptors, often referred to as cyclophilins (CYPs), and the FK506- and rapamycin-binding proteins (FKBPs). Plant immunophilins were previ- ously shown to be involved in the function of innate immunity in higher plants (Aumuller et al., 2010). Immunophilins are a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (PPiase) (Fisher Gene xxx (2014) xxx–xxx Abbreviations: PAMPs, pathogen-associated molecular patterns; PRRs, pattern recog- nition receptors; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; CYPs, cyclophilins; FKBPs, rapamycin-binding proteins; PPiase, peptidyl-prolyl cis-trans isomerase activity; cDNA, complementary DNA; qRT-PCR, quantitative real-time PCR; LB, Luria–Bertani; X-Gluc, 5-bromo-4-chloro-3-indolyl-b-D-Glucuronide; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; GFP, green fluorescent protein; GUS, beta-glucuronidase; dpi, days post inoculation; PTI, Pattern Triggered Immunity; ETI, Effector Triggered Immunity; MEKK1, MAPK/ERK kinase kinase member A1; EDS1, ENHANCED DISEASE SUSCEPTIBILITY 1; PR1, PATHOGENESIS-RELATED 1; PAD4, PHYTOALEXIN DEFICIENT 4; WRKY33, WRKY DNA-BINDING PROTEIN 33; bGS2, β-GLUCAN SYNTHASE 2; NADPH, Nicotinamide Adenine Dinucleotide Phosphate Oxidase; SA, salycilic acid; NLS, Nuclear Localization Signal. ⁎ Corresponding author at: 219 Bessey Hall, Department of Plant Pathology and Microbiology, Iowa State University, Ames 50014, IA, USA. Tel.: +1 515 294 3120. E-mail addresses: gennady@iastate.edu, gpogorelko@yandex.ru (G.V. Pogorelko), marja-2007@yandex.ru (M. Mokryakova), oksfursova@yandex.ru (O.V. Fursova), insaz@yandex.ru (I. Abdeeva), eleopiru@vigg.ru (E.S. Piruzian), sergey.bruskin@gmail.com (S.A. Bruskin). GENE-39399; No. of pages: 11; 4C: 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.029 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Please cite this article as: Pogorelko, G.V., et al., Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defense response against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.029