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Gene xxx (2014) xxx–xxx
GENE-39399; No. of pages: 11; 4C:
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Characterization of three Arabidopsis thaliana immunophilin genesinvolved 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, USAb NI Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow 119991, Russiac Geocryology Department, Moscow State University, Leninskie Gory 1, Moscow 119992, Russia
Abbreviations: PAMPs, pathogen-associated moleculanition receptors; ROS, reactive oxygen species; MAPK, miCYPs, cyclophilins; FKBPs, rapamycin-binding proteins; Pisomerase activity; cDNA, complementary DNA; qRT-PCRLuria–Bertani; X-Gluc, 5-bromo-4-chloro-3-indolyl-b-D-Gdodecyl sulfate polyacrylamide gel electrophoresis; GFP,beta-glucuronidase; dpi, days post inoculation; PTI, PatEffector Triggered Immunity; MEKK1, MAPK/ERK kinENHANCED DISEASE SUSCEPTIBILITY 1; PR1, PATHPHYTOALEXIN DEFICIENT 4;WRKY33, WRKY DNA-BINDINSYNTHASE 2; NADPH, Nicotinamide Adenine Dinucleotideacid; NLS, Nuclear Localization Signal.⁎ Corresponding author at: 219 Bessey Hall, Depar
Microbiology, Iowa State University, Ames 50014, IA, USAE-mail addresses: [email protected], gpogorelko@y
[email protected] (M. Mokryakova), oksfursova@[email protected] (I. Abdeeva), [email protected] (E.S. Piruz(S.A. Bruskin).
0378-1119/$ – see front matter © 2014 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2014.01.029
Please cite this article as: Pogorelko, G.V., etresponse against Pseudomonas syringae, Gen
a b s t r a c t
a r t i c l e i n f oArticle history:Accepted 10 January 2014Available online xxxx
Keywords:Immunophilin familyPlant–pathogen interactionPlant immune system
Plant immunophilins are a broadly conserved family of proteins, which carry out a variety of cellular functions. Inthis study, we investigated three immunophilin genes involved in theArabidopsis thaliana response to Pseudomo-nas syringae infection: a cytoplasmic localized AtCYP19, a cytoplasmic and nuclear localized AtCYP57, and onenucleus directed FKBP known as AtFKBP65. Arabidopsis knock-out mutations in these immunophilins result inan increased susceptibility to P. syringae, whereas overexpression of these genes alters the transcription profileof 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. AtCYP19was shown to be involvedin reactive oxygen species production, and both AtCYP57 and AtFKBP65 provided callose accumulation in plantcell wall. Identification of the involvement of these genes in biotic stress response brings a new set of data thatwill 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 impactson the growth and development of plants, including crop species. Inorder to survive, plants have evolved a sophisticated defense systemagainst a variety of pathogens, including viruses, bacteria, fungi, andnematodes (Hou et al., 2009). Stress is recognized and transmitted bythe signal transduction system which influences the regulatory ele-ments of stress-inducible genes involved in retrograde signaling to thespecific genes and proteins that provide stress resistance (Chen et al.,2002; Knight and Knight, 2001). Studying the functions of stress-
r patterns; PRRs, pattern recog-togen-activated protein kinase;Piase, peptidyl-prolyl cis-trans, quantitative real-time PCR; LB,lucuronide; SDS-PAGE, sodiumgreen fluorescent protein; GUS,tern Triggered Immunity; ETI,ase kinase member A1; EDS1,OGENESIS-RELATED 1; PAD4,G PROTEIN 33; bGS2, β-GLUCANPhosphate Oxidase; SA, salycilic
tment of Plant Pathology and. Tel.: +1 515 294 3120.andex.ru (G.V. Pogorelko),ndex.ru (O.V. Fursova),ian), [email protected]
ights reserved.
al., Characterization of three Ae (2014), http://dx.doi.org/10
inducible genes enables an understanding of the underlying mecha-nisms of plant–stress interactions and modulation of their function bymolecular 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 microbialmolecules named pathogen-associated molecular patterns (PAMPs).Specific PAMPs are detected via corresponding trans-membranepattern recognition receptors (PRRs) and initiate intracellular immuneresponses (Zipfel, 2008). These responses include the generation ofreactive oxygen species (ROS), protein phosphorylation (Gimenez-Ibanez and Rathjen, 2010), and downstream activation of signalingcascades. Mitogen-activated protein kinase (MAPK) signaling plays acentral role in plant response against pathogen intrusion. This pathwaystarts with initiation by a PRR responsive gene called MEKK whichprovides a downstream MAP kinase signaling that leads to specificpatterns of stress-responsive gene expression as well as changes inpost-translational modifications.
The immunophilin protein family functions as receptors forimmunosuppressive drugs, and has been found in a broad range oforganisms, including bacteria, fungi, animals, and plants. Two groupsof immunophilin receptors exist in plants: cyclosporin A receptors,often referred to as cyclophilins (CYPs), and the FK506- andrapamycin-binding proteins (FKBPs). Plant immunophilins were previ-ously shown to be involved in the function of innate immunity in higherplants (Aumuller et al., 2010). Immunophilins are a family of enzymeswith a peptidyl-prolyl cis-trans isomerase activity (PPiase) (Fisher
rabidopsis thaliana immunophilin genes involved in the plant defense.1016/j.gene.2014.01.029
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et al., 1989; Schreiber, 1991) and are involved in the folding of targetproteins (Hur and Bruice, 2002; Schiene-Fischer and Yu, 2001; Shaw,2002; Vespa et al., 2004) and the determination of their structure(Reimer and Fischer, 2002). Yeast Cyclophilin A, besides controllingprotein folding, has been shown to localize in the cell nucleus and is in-volved in the control of meiosis (Arevalo-Rodriguez et al., 2004). Anumber of publications reveal the involvement of plant immunophilinsmostly in response to different types of abiotic but there are also someevidences of its participation in biotic stresses response control (Ahnet al., 2010; Aviezer-Hagai et al., 2007; Kumari et al., 2012; Nigamet al., 2008). Immunophilins can act as molecular effector switches(Coaker et al., 2006) in the control of thermotolerance (Cho et al.,2005; Kiełbowicz-Matuk et al., 2007; Meiri et al., 2010), draught andsalt stress (Chen et al., 2007; Hajheidari et al., 2005; Karali et al., 2012;Liu et al., 2009; Mahfouz et al., 2006), acid stress bymaintaining pH ho-meostasis (Bissoli et al., 2012), and oxidative stress (Laxa et al., 2007).Pigeonpea cyclophilin expressed in Arabidopsis plants has been shownto confer tolerance tomultiple abiotic stresses andwas found to be pre-dominantly localized in the nucleus, where it is thought to directly reg-ulate various stress-responsive genes (Sekhar et al., 2010).
Plant peptidyl-prolyl cis-trans isomerase AtCYP19-1 cyclophilinencoded by At2g16600, also known as ROC3, has a suggested role inseed development (Stangeland et al., 2005), but is also expressed inseedlings, stems, and leaves (Chou and Gasser, 1997). At5g48570,which is also known as ROF2 (AtFKBP65), encodes a heat stress proteinthat participates in long term acquired thermotolerance, which is simi-lar to the homologous ROF1 (AtFKBP62) gene that controls modulationof the heat shock transcription factor HsfA2 (Meiri et al., 2010). TheAt4g33060 (AtCYP57) cyclophilin gene is currently only classified byits sequence (He et al., 2004), and its function has still not beeninvestigated.
In this study, we elucidate the role of two plant cyclophilin typegenes, AtCYP19 and AtCYP57, and the AtFKBP65 FKBP type gene in bioticstress responses, with an emphasis on plant resistance to pathogenattack.
2. Materials and methods
2.1. Constructs for expression of AtCYP19, AtCYP57 and AtFKBP65 nativeand tagged genes and for promoter analysis
Total RNA from Arabidopsis thaliana leaves infectedwith Pseudomonassyringaewas extracted using an SV Total RNA Isolation System (Promega,Z3101) andfirst strand cDNA synthesiswas doneusing the SuperScript IIIFirst-Strand Synthesis System (Invitrogen, 18080051) following themanufacturer's recommendations.
AtCYP19 cDNAwas amplifiedwith the use of primers: AtCYP19_Forwand AtCYP19_Rev, contains EcoRI and BamHI sites respectively (Supple-mentary Data 1 for primer sequences). Primers AtCYP57_Forw andAtCYP57_Rev containing KpnI and ClaI sites were used to amplifyAtCYP57 cDNA and AtFKBP65_Forw and AtFKBP65_Rev with incorporat-ed XhoI and BamHI restriction enzyme sites were used for AtFKBP65cDNA amplification. cDNA PCR fragments were obtained by the use ofEncyclo DNA polymerase with proof reading 3′-5′ exonuclease activity(Evrogen, PK001) and cloned into a pGEM-T Easy vector (Promega,A1360). To create the expression cassette, pGEM-T Easy recombinantvectors were treated with restriction enzymes appropriate to the sitesincorporated into the primers, and the DNA fragments were clonedinto pKANNIBAL vector (Wesley et al., 2001) treated with the same en-zymes: EcoRI and BamHI for AtCYP19, KpnI and ClaI for AtCYP57, XhoIand BamHI for AtFKBP65. Recombinant pKANNIBAL vectors were thendigested with NotI enzyme and DNA fragments containing cDNA ofeach gene under the control of the 35S promoter were cloned intopMLBart binary vector (Gleave, 1992), also digested with NotI.
To fuse immunophilin geneswithGFP coding sequence primers GFP-F1 and GFP-R1, both containing BamHI sites or GFP-F2 and GFP-R2 with
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
ClaI sites were used to amplify internal GFP sequence from pSMGFPplasmid available from ABRC DNA stock center (Cat. No. CD3-326,www.arabidopsis.org). Obtained PCR fragments were cloned topGEM-T Easy vector and then cut with appropriate enzyme toclone it into pKANNIBAL vector containing AtCYP19, AtFKBP65(treated with BamHI) and AtCYP57 (treated with ClaI) followed bycloning expression cassette into pMLBart binary vector pre-treatedwith NotI.
Promoter sequences of the AtCYP19, AtCYP57 and AtFKBP65genes were amplified from Arabidopsis Columbia-0 ecotype total ge-nomic DNA extracted using cetyl-trimethylammonium bromide(CTAB) isolation buffer according to the protocol of Stewart and Via(1993). The following primer sets were used: AtCYP19_Prom_Forw andAtCYP19_Prom_Rev; AtCYP57_Prom_Forw and AtCYP57_Prom_Rev;and AtFKBP65_Prom_Forw and AtFKBP65_Prom_Rev. All forwardprimers for promoter amplification contained NotI restriction sites andall reverse primers had EcoRI sites. PCR fragmentswere cutwith EcoRI en-zyme and ligated with the GUS gene including a transcriptional termina-tor PCR fragment treated also with EcoRI. To amplify the GUS genetogether with the NOS terminator, GUS_Forw and GUS_Rev primers con-taining EcoRI and NotI restriction sites, respectively, were used. The pLD3binary vector was used as a source of the uidA beta-glucuronidase GUSgene sequence (GenBank ID: S69414.1). Products of ligation were usedfor the second round of amplification with the use of promoter specificforward primer and a GUS specific reverse primer. Hybrid promoter-GUS PCR fragments were digested directly with the NotI enzyme andcloned into the pMLBart binary vector also pre-treated with NotI.
For transient expression of immunophilins-GFP fusions controlledby native promoters in Nicotiana benthamiana plants, immunophilinspromoters were amplified with the use of primers listed above but con-taining SacI restriction enzyme site on forward primers and XhoI site onreverse. Treatment with these enzymes allowed replacing CaMV35Spromoter in pKANNIBAL vector (containing immunophilin-GFP fusioncassette) with immunophilin native promoters followed by cloningexpression cassette into pMLBart binary vector pre-treated with NotI.
2.2. Plant material. Transformation of Arabidopsis and agroinfiltration ofN. benthamiana leaves
Obtained binary vectors containing genes under CaMV35S promoterwere transformed into Agrobacterium tumefaciens C58 cells by electro-poration, and Arabidopsis plants were transfected with these cells bythe floral-dip method (Clough and Bent, 1998).
The abaxial leaf surface of 6-week-oldN. benthamiana plants was in-filtrated with 106 cfu/mL Agrobacterium culture in infiltration buffer(10 mM MES, 10 mM MgCl2, pH = 5.8) as was described previouslyusing a syringe without a needle (Swanson et al., 1988). AgrobacteriumC58-Z707 strain (Kanamycin resistant) was used for co-infection withP. syringae (Pto DC3000).
2.3. Inoculations and disease development
For plant treatment, bacterial culture of Pseudomonas syringae pv.tomatowere grown overnight in LB medium at 25 °C up to OD600 = 1.Cells were collected by centrifugation at 4000 rpm for 10 min and re-suspended in infiltration buffer (10 mM MES, 10 mM MgCl2, pH =5.8) and their concentrations were adjusted to 106 cfu/mL. Preparedcell suspensions were then pressure infiltrated into the abaxial side ofArabidopsis overexpressing transgenic plants or knock-out mutantleaves lacking expression of AtCYP19 (SALK_063724), AtCYP57(SAIL_621_H11), and AtFKBP65 (SAIL_355_A02) obtained from ABRCseed stock collection (http://abrc.osu.edu/).
ForN. benthamiana treatment in three days after infiltrationwith theAgrobacterium cultures carrying one of each of the 35S:AtCYP19-GFP,35S:AtCYP57-GFP, and 35S:AtFKBP65-GFP gene expression cassettes, abacterial suspension of P. syringae was then also pressure infiltrated
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into the abaxial side of both wild type and agroinfiltrated leaves. Toassay bacterial growth, a circle of 1 cm in diameter was cut from theleaf keeping injection point in center.
2.4. cDNA preparation and real-time qPCR
Total RNAs were isolated from plant tissues using Trizol reagent(Invitrogen) and treatedwithDNase I (Invitrogen) for 20min to removepossible contaminating genomic DNA. Total RNAwas assessed on a 0.7%agarose gel, quantified and normalized by NanoDrop ND-1000 spectro-photometer. For qRT-PCR analysis, first-strand cDNAswere synthesizedfrom 3 μg of total RNA using Superscript III reverse transcriptase(Invitrogen) according to the manufacturer's recommendations. Real-time qPCR was performed on an iCycler Real-Time system (Bio-Rad).Each reaction was done in a final volume of 25 μL containing 12.5 μLof SYBR Green Master Mix reagent (Fermentas), 2.0 μL of cDNA sam-ple, and 200 nM gene-specific primers, designed to generate a size of150–200 bp products using Vector NTI 9.0 software (Informax). TheqPCR conditions were as follows: 50 °C for 2 min, 95 °C for 10 min,45 cycles of 95 °C for 15 s and 60 °C for 30 s and 72 °C for 45 s. Atthe end of the 45 cycles, a melting curve was generated to analyzethe specificity of the reactions. Each cDNA sample was tested inthree replicates. Expression level of the Actin 2 gene was used asthe endogenous control. The relative expression level was calculatedas 2−ΔΔCT [ΔCT = CT, gene of interest − CT, Actin 2. ΔΔCT = ΔCT, treatment
− ΔCT, Col-0].Absolute gene transcript numbers were quantified as described
previously (Lu et al., 2012).
2.5. In planta growth and disease development
All measurements were taken from rosette leaves of 5–6-week-oldArabidopsis plants and from fourth and fifth leaf from the base of theN. benthamiana plant. To determine bacterial growth a piece of leaf(30 mm2) was ground in 500 μL of 10 mMMgCl2 solution and a seriesof dilutions from 10− up to 10−8, were plated on LB media containing50 mg/L of Rifampicin. Calculations were done from the dilution thatgave approximately 50–70 colonies.
Flg22 peptide was purchased from EZBiolab Inc. (Westfield, IN,USA). Indicated concentration of flg22 solution was applied to leavesof 4 to 5 week-old plants by infiltration using a needleless syringe oneday before infiltration with bacteria.
2.6. Determination of Callose Deposition and Hydrogen PeroxideAccumulation. Light, Fluorescence, and Confocal microscopy
For hydrogen peroxide visualization, leaves were cut from 5-week-old plants and dipped for 16 h in a solution containing1 mg/mL 3,3′-diaminobenzidine, pH 5.0. Chlorophyll was extractedwith hot 96% ethanol, and the leaves were photographed with aCanon EOS300D camera.
For callose examination two more expanded leaves from six inde-pendent 5-week-old plants were cleared with 96% ethanol and fixedin an acetic acid:ethanol (1:3) solution for 2 h, sequentially incubatedfor 15 min in 75% ethanol, in 50% ethanol, and in 150 mM phosphatebuffer, pH 8.0, and then stained for 2 h at 25 °C in 150 mM phosphatebuffer, pH 8.0, containing 0.01% (w/v) aniline blue. After staining, leaveswere examined by UV filter (excitation wavelength = 330–380) usingthe fluorescence microscope Leica DMIRE2 microscope.
N. benthamiana leaves transiently expressing the immunophilin-GFP-fusion were examined with a Carl Zeiss LSM 510 META confocalmicroscope, using 488 nm excitation and 520–550 nm emissionwavelengths for GFP.
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
2.7. GUS expression analysis
The histochemical localization of GUS in transfected N. benthamianaleaves was performed as described previously (Jefferson et al., 1987).Five-week-old N. benthamiana leaves were fully infiltrated withAgrobacterium cultures to provide expression of AtCYP19, AtCYP57and AtFKBP65 promoter GUS fusions, and three days later a smallvolume of P. syringae culture inoculum was injected into the center ofthe same leaves in an approximately 5–10 mm2 area. Twentyfour hours after pathogen injection, fresh leaves were collected and im-mersed in a histochemical reactionmixture containing 1 mg/mLX-Gluc(5-bromo-4-chloro-3-indolyl-b-D-Glucuronide; Duchefa) in 150 mMpotassium phosphate buffer pH 7.0, 10 mM potassium ferrocyanide,10 mM potassium ferricyanide and 0.05% Triton X-100. The histochem-ical reaction was performed in the dark at 37 °C for 24 h. Stained leaveswere incubated in 70% ethanol for 48 h to remove chlorophyll prior totaking pictures.
2.8. Western blot analysis
Immunoblotting analysis was performed according to Magi andLiberatori (2005). Twenty micrograms of total protein was loaded perwell for 10% SDS-PAGE. After separation, the proteins were electropho-retically transferred to a Trans-Blot Transfer Medium membrane (Bio-Rad, 162-0112) using the Trans-Blot Cell (Bio-Rad) with a standardtransfer buffer. Primary Anti-GFP monoclonal antibody (Covance, CA,USA, MMS-118P) at a dilution of 1:4000 and secondary Anti-MouseIgG (wholemolecule)-Peroxidase antibody (Sigma, A9044) at a dilutionof 1:30,000 were used for the detection of GFP-fusion proteins.Membranes were treated with the HyGlo Quick Spray Reagent B forperoxidase activity (Denville, E2400) and immediately visualized byChemiDoc™ XRS+ System (BioRad, 170-8265).
3. Results
3.1. In silico analysis of the A. thaliana immunophilin superfamily:searching for genes involved in plant-microbe interactions
To study the function of plant immunophilins and their complex func-tions in plant–pathogen interactions, we performed a meta-analysison available gene expression data for Arabidopsis immunophilinsduring response to biotic stresses. With the help of the software toolGenevestigator V3 (www.genevestigator.ethz.ch), and public microarraydatabases of Arabidopsis genes, we analyzed the expression changes of52 genes characterized as Arabidopsis immunophilins in 201 independentmicroarray experimentswhich investigated gene expressionduringbioticand abiotic stresses. We used a 2-fold change in gene expression as athreshold value for determining a systemic change in expression patterns.Using these parameters we identified 32 Arabidopsis immunophilingenes whose expression increased more than 2-fold, representingimmunophilins which are involved in plant responses to stress factors.To identify those genes whose expression changed specifically duringbiotic stress, we analyzed seventeen different microarray experimentsperformed on plant–pathogen interactions. Our data analysis showedthat 5 Arabidopsis immunophilin genes increased their expression, while19 genes reduced their expression under biotic stress conditions. Thus,there is a reduced expression trend for a majority of immunophilingenes when exposed to biotic stress.
To investigate the function of specific immunophilins that are in-volved in the Arabidopsis–pathogen interactions, we chose the threegenes that showed significantly increased expression only during re-sponses to pathogens. The AtFKBP65 (product-peptidyl-prolyl cis-transisomerase), an alleged FK506-binding protein whose expression in-creased 17.69 times during interaction with Bemisia tabaci type B, 5.91times with Heterodera schachtii, 9.16 times with P. syringae (avrRpm1),and 4.52 times with P. syringae (avrRps4); the ROC3 (AtCYP19)
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Please cite this article as: Pogorelko, G.V., et al., Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defenseresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.029
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cyclophilin – a short gene lacking other functional domains besideconserved CYP and has been described as a gene which might be in-directly involved in abiotic stress response due to participation inplant growth and development (Yang et al., 2009), and whose ex-pression is increased 2.94 times by the action of P. syringae; andthe AtCYP57 gene, a cyclophilin-type family protein, whose expres-sion is increased 5.64 times by P. syringae (avrRpm1). We hypothe-sized that these genes activate components of the plant defensesystem.
3.2. Expression of AtCYP19, AtCYP57 and AtFKBP65 is activated duringpathogen invasion
To confirm our meta-analysis data, we used quantitative RT-PCR tocheck the expression of AtCYP19, AtCYP57 and AtFKBP65 in A. thalianaplants infected with P. syringae pv. tomato (Fig. 1A).
Threeweek old Arabidopsisplantswere dip inoculatedwith bacterialconcentrations of 106 colony forming units per milliliter (cfu/mL), andRNA samples were extracted from leaves at different time points post-infiltration. RT-PCR analysis showed significantly increased expressionof all 3 genes after infection relative to controls (more than 10-folds at6 dpi).
3.3. Promoters of the AtCYP19, AtCYP57 and AtFKBP65 genes activateexpression locally in leaf tissues invaded by pathogenic bacteria
To examine whether AtCYP19, AtCYP57 and AtFKBP65 proteinsact locally in plant tissues or provide signaling across whole organs,we analyzed expression of the endogenous promoters of AtCYP19,AtCYP57, and AtFKBP65. 475, 429 and 449 upstream nucleotides ofAtCYP19, AtCYP57, and AtFKBP65 promoter regions were fused withreporter gene. Using the Softberry online bioinformatics tools(http://linux1.softberry.com/berry.phtml) allowed us to identify en-dogenous promoter elements 58 nucleotides upstream of AtCYP19start codon, 214 nucleotides upstream AtCYP57 and 303 nucleotides up-streamAtFKBP65. Each identified promoter regionwas cloned and fusedto the beta-glucuronidase (GUS) coding sequence to produce T-DNAreporter constructs.
The entire surface areas of N. benthamiana leaves were inoculatedwith A. tumefaciens cell cultures carrying one of each of the 3 constructs(Imm:Prom:GUS, Supplementary Fig. 1), and 3 dpi cell suspensions ofP. syringaewere separately infiltrated onto single leaveswith an infiltra-tion diameter of 6–10mm.We detected activation of GUS expression inall of the samples, which showed that all three of the investigatedimmunophilin genes are induced during response to the P. syringaeinfection (Figs. 1B1–B7).
3.4. AtCYP19, AtCYP57 and AtFKBP65 knock-out mutants are moresusceptible to P. syringae infection and have defects in the PAMP-inducedsignaling pathway
To examine whether the lack of expression of AtCYP19, AtCYP57 andAtFKBP65 genes causes any changes in plant–microbe interactions,knock-out plants for each gene were analyzed. Knock-out mutationswere confirmed by quantitative real-time PCR (Fig. 2A). The susceptibil-ity of knock-out plants was assessed by inoculating leaves withP. syringae and subsequently evaluating bacterial growth rate at
Fig. 1. (A) Expression pattern of AtCYP19, AtCYP57, and AtFKBP65 under biotic stress. P. syringaeand relative gene expression level was estimated by qRT-PCR on three independent inoculatiofiltration. The comparative threshold cycle method was used for determining differences betwmean ± SD. Asterisks indicate significant differences between infiltrated and wild type plants,under the AtCYP19 promoter (B1), the AtCYP57 promoter (B2), and the AtFKBP65 promoterAgrobacterium cell cultures harboring expression constructs of immunophilin promoter:GUS fusents mock inoculation of P. syringae 106 cfu/mL cell suspension to the leaf in three days after ipromoter, and B5, B6, B7, represents inoculationwith Agrobacterium cell culture carrying emptythe AtFKBP65 promoter (B7) with the absence of P. syringae.
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
different time points post-inoculation. At 1–6 days post-inoculation(dpi) all mutant plants displayed significantly enhanced P. syringaegrowth compared to wild-type (Fig. 2B).
Since P. syringae pv. tomato can induce both Pattern Triggered Im-munity (PTI) and Effector Triggered Immunity (ETI) (Chang and Nick,2012; Zhang et al., 2010), We tested the effect these immunophilinknock-out mutations have on the Arabidopsis response to P. syringaeafter being induced by purified flg22 (Fig. 2C) (Chang and Nick, 2012).We determined significant 1.6-fold, 1.7-fold, and 1.5-fold decrease ofP. syringae growth in treatedwith flg22 leaves comparing tomock treat-edwhereas Col-0 plants showed 2-fold difference between induced andnon-induced PTI revealing defects in PAMP-induced signal transductionpathway.
3.5. Transgenic Arabidopsis plants overexpressing AtCYP19, AtCYP57 andAtFKBP65 are more resistant to P. syringae
To determine whether increased expression of the three selectedgenes affects plant susceptibility to bacterial invasion, we transformedwild-type Arabidopsis plants with an expression cassette containingeach individual immunophilin gene under the control of the CaMV35Spromoter (35S:Imm, Supplementary Fig. 1). Using RT-PCR we wereable to confirm that the transgenic plants express the immunophilingenes 6.5–11 fold higher than wild-type plants (Fig. 2E). To analyzethe effect, the over expression of these genes has on the plant-pathogen interaction, each transgenic line was inoculated withP. syringae. By 2 and 3 dpi all transgenic lines showed a significantly in-creased resistance to P. syringae (Fig. 2F). Lines expressing AtCYP19,AtCYP57, and AtFKBP65 showed a significant reduction in P. syringaegrowth of 40%, 15%, and 28% respectively compared to growth onwild-type controls (Fig. 2D). Similar results of P. syringae growthsuppression were observed using transiently expressing of Imm-GFPconstructs N. benthamiana tissues (Supplementary Fig. 2).
3.6. Increased expression of immunophilins leads to changes in pathogen-responsive gene expression providing callose deposition and hydrogenperoxide accumulation
Reduced susceptibility to pathogens can be caused by the direct ac-tion of certain host proteins against microbial mechanical or enzymaticpenetration into host cells, or by enhanced pathogen perception andsubsequent activation of complex immune responses. To determinewhether overexpression of immunophilins alters the transcriptionof pathogen responsive genes in the absence of a pathogen, we usedRT-PCR to measure the relative expression levels of six major pathogenrelated genes known to be involved in the primary Arabidopsis responseto P. syringae infection (MAPK/ERK kinase kinase member A1 (MEKK1),PHYTOALEXIN DEFICIENT 4 (PAD4), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), PATHOGENESIS-RELATED 1 (PR1), WRKY DNA-BINDINGPROTEIN 33 (WRKY33), and β-GLUCAN SYNTHASE 2 (bGS2)) (Fig. 3A).MEKK1 expression was found to be 7.6 fold higher in 35S:AtCYP19plants relative to wild-type. 35S:AtCYP57 lines showed 21, 6.7, 6.1,and 15.6 fold increases in the expression level of PAD4, PR1, WRKY33,and bGS2 genes respectively. And 35S:AtFKBP65 plants displayed 11.2and 8.4 folds higher level of WRKY33 and bGS2 genes.
To determine whether the reduced susceptibility to pathogens ob-served in transgenic plants is due to the activation of defense responses,
(Ps) cell culture (106 cfu/mL) was infiltrated into leaves of 6-week-old Arabidopsis plantsns. Total RNA was extracted from infected leaves at 0 h, 1 day, 4 days, and 6 days after in-een transcript copy numbers in infected and non-infected plants. All data represents theaccording to Student's t-test (n= 3; P b 0.05). (B1–B7) Activation of GUS gene expression(B3) after local injection of P. syringae 106 cfu/mL cell suspension in three days aftersion were infiltrated to the same leaves, resulting in a 6–10 mm infection zone. B4 repre-nfiltration with Agrobacterium cell culture harboring empty vector with GUS gene withoutvector with only GUS gene under AtCYP19 promoter (B5), the AtCYP57 promoter (B6) and
rabidopsis thaliana immunophilin genes involved in the plant defense.1016/j.gene.2014.01.029
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Please cite this article as: Pogorelko, G.V., et al., Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defenseresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.01.029
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callose deposition andhydrogen peroxide (H2O2) accumulation in unin-fected immunophilin overexpressing plants was compared with wild-type (Figs. 3B and C). High callose accumulation was detected in 35S:AtCYP57 plants and significantly more callose deposition was observedin 35S:AtFKBP line compared to wild-type, which positively correlateswith the level of bGS2 expression (Fig. 3A). No callose deposition wasdetected in the leaf tissues of 35S:AtCYP19 lines, however, it showed adramatic accumulation of H2O2.
Retrograde activation of bGS2 led to the accumulation of callose in35S:AtCYP57 and 35S:AtFKBP65 lines. Furthermore, our qualitativeobservations indicate that this accumulation may positively correlateswith the level of bGS2 expression (Figs. 3A and B).
3.7. Localization of transiently expressed of AtCYP19-GFP, AtCYP57-GFP,and AtFKBP65-GFP proteins in N. benthamiana under the native promotersin presence of P. syringae
To gain insight into the mechanism of immunophilin induced resis-tance, we analyzed the sub-cellular localization of three selectedimmunophilins proteins fused with Green Fluorescent Protein (GFP)expressed using their native promoters. GFP was fused to the 3′-endof the coding sequence of each gene, which translated into immunophilinproteins with C-terminal GFP tags (ImmProm:Imm:GFP, SupplementaryFig. 1). The immunophilin:GFP expression cassettes were infiltrated intoN. benthamiana leaves and were co-infiltrated with Pseudomonas 3 dayslater. After three days of bacterial interrogation the same leaves were an-alyzed using confocalmicroscopy to localize the expression of each fusionprotein.
The immunophilin AtCYP19 gene encodes a protein with a singleconserved domain (CYP) (He et al., 2004). The AtCYP57protein containsin addition to the CYP domain an Arg/Lys rich domain with two nucleartargeting signals within it (He et al., 2004). AtFKBP65 has originallybeen described as a gene coding a protein consisting of three repeatsof the FKBP domain, three tetratricopeptide repeats and a calmodulin-bindingmotif, which aremore commonly found in cytoplasmic proteins(He et al., 2004).
After analyzing the infiltrated N. benthamiana leaves we found thatthe immunophilin genes showed different subcellular localizations.The AtCYP19-GFP fusion protein localized to the cytoplasm in planta(Figs. 4A1–A4). While the AtCYP57-GFP fusion protein was localizedin the cell nucleus as well as the cell cytoplasm (Figs. 4B1–B4). Finally,The AtFKBP65-GFP was found to be localized specifically in the cellnucleus (Figs. 4C1–C4). Interestingly, no nuclear localization signal se-quences could be identifiedwithin this protein using latest bioinformat-ics tools. However, AtFKBP65 was previously shown to be localized incell nucleus upon heat stress (Meiri et al., 2010). Protein expression ininfiltrated leaves was checked by Western immuno blotting in orderto verify that signal was detected from full protein fusions but notfrom truncated versions (Figs. 4A4, B4, C4). To exclude the presence ofsmall truncated parts of GFP in infiltrated tissues experiment wasrepeated 2 times using Novex® 4–20% gradient tris-glycine gels andlong time of membrane over-exposure during signal visualization andonly single signals referring to full fusion proteins were detected.
Co-infiltration of N. benthamiana leaves using immunophilin:GFPfusion constructs and Pseudomonas cell cultures revealed a decrease inthe bacteria reproduction relative to empty vector controls. The degree
Fig. 2. (A) Expression level of AtCYP19, AtCYP57, and AtFKBP65 in 6-week-old knock-out mutan(KO) and Col-0 plants to P. syringae. Mock treatment resulted in zero colonies and is not presenCol-0 plantswith induced PTI to P. syringae. Bacterial culture (106 cfu/mL)was inoculated into th(+). The bacterial numberwasmeasured at 5 dpi. (D)Observed hypersensitive response phenoto Col-0 plants at 5 days post infection with P. syringae (106 cfu/mL). (E) The absolute quantArabidopsis transgenic plants. (F) Growth of P. syringae in transgenic Arabidopsis lines overexprBacteriawere infiltrated at 106 cfu/mL and isolated from leaves at the indicated time points aftenormalizations in qRT-PCRwere done byActin-2 gene expression. The Y axis is the scale of exprebetween transcript copy numbers inwild-type and transgenic plants. All infection data points rEach point representsmean± standard deviation. Asterisks indicate significant differences betw
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
of bacterial growth reduction positively correlatedwith accumulation ofectopic gene transcripts and the amount of observedHR (Supplementa-ry Fig. 2). Starting from 24 h post-inoculation, bacterial growth onAtCYP19-GFP and AtFKBP65-GFP transformed tissues was determinedto be significantly less then mock inoculated tissues.
4. Discussion
In this study, we have chosen to focus on three immunophilin genesthat show significant up-regulation during the responses of Arabidopsisto biotic stress. To gain insight into the function of these threeimmunophilins during pathogen invasion, we characterized the effectsthat knock-out and overexpression mutations in these genes have onthe response of Arabidopsis to bacterial infection. Furthermore, we ec-topically expressed these genes in N. benthamiana leaves, to test theireffects in a different pathosystem. The increased susceptibility of theArabidopsis knock-out mutants to Pseudomonas infection togetherwith the experimentally confirmed responses of AtCYP19, AtCYP57 andAtFKBP65 transcript levels in previous microarray data strongly sup-ports the involvement of these genes in the plant response to pathogeninfection. Furthermore, overexpressionof the individual immunophilinsled to the suppression of P. syringae colonization in all three transgenicArabidopsis lines as well asN. benthamiana leaves ectopically expressingAtCYP19-GFP and AtFKBP65-GFP. Additionally, we detected a non-significant resistance trend to P. syringae on N. benthamiana leaves ec-topically expressing AtCYP57-GFP construct (Supplementary Fig. 2).We suggest that this reduced efficacy could be caused by differencesin the host specific response in tobacco plants, or the influence of GFPprotein on the natural activity of AtCYP57.
Constitutive expression of AtCYP19 resulted in increased accumula-tion of reactive oxygen species (ROS) (Fig. 3C), which inhibit pathogengrowth by acting as a bactericidal agent, inducing cell wall modifica-tions, activating pathogen related genes, and activating salicylic acidbiosynthesis (Bradley et al., 1992; Desikan et al., 2001; Leon et al.,1995; Peng andKuc, 1992). ROS can be produced by cellwall, mitochon-dria, plastids, endoplasmic reticulum, peroxisomes, NADPHoxidaseme-tabolisms and as a consequence of cell membrane electron transporting(Baxter-Burrell et al., 2002; Sharma et al., 2012; Torres et al., 2001,2006). Indeed, ROS were proposed to act as a primary signaling mole-cules in abiotic and biotic stresses, activatingmitogen-activated proteinkinase (MAPK) pathways through the induction of specific kinases(Kovtun et al., 2000). In our study, AtCYP19 expressing plants showedincreased expression ofMEKK1 (Fig. 3A), which is known to play a cen-tral role in MAPK pathways which respond to pathogen attack (Hamelet al., 2012), and its activity is regulated by ROS (Nakagami et al., 2006).
The increased expression level of MEKK1 in 35S:AtCYP19 plants to-gether with the absence of up-regulation PR1 gene – a salycilic acid(SA) pathway marker gene (Chen et al., 1993) – indicates that thisgene may be involved in SA-independent activation of the MAPK cas-cade. AtCYP19 is localized in cytoplasm (Figs. 4A1–A3) and presumablyinteracts with cytosolic ROS producing proteins and particularly withNADPH oxidase or its regulating factors. In contrast to mammal'smultiprotein NADPH oxidase complex (Cross and Segal, 2004) plantNADPH oxidases are single cytosolic proteins (Davis et al., 1998)which have not been well characterized. We propose AtCYP19 may in-teract with NADPH oxidase or act as upstream regulator to produce
ts examined by qRT-PCR. (B) Susceptibility of Arabidopsis 6-week-old knock-out mutantsted on a graph. (C) Susceptibility of Arabidopsis 5-week-old knock-out mutants (KO) ande same knock-outmutants leaves one day after pretreatmentwithwater (−) or 1 μMflg22type onArabidopsis leaves of knock-out (KO) and overexpressing (35S) lines in comparisonifications of AtCYP19, AtCYP57, and AtFKBP65 transcript accumulation in overexpressingessing (35S) AtCYP19, AtCYP57, and AtFKBP65 and wild type plants after hand infiltration.r inoculation.Mock treatment resulted in zero colonies and is not presented on a graph. Allssion level. The comparative threshold cyclemethodwas used for determining differencesepresent the average of three biological replicates for each of the three technical replicates.een transgenic plants andwild type plants, according to Student's t-test (n= 3; P b 0.05).
rabidopsis thaliana immunophilin genes involved in the plant defense.1016/j.gene.2014.01.029
Fig. 3. (A) qRT-PCR analysis of selected pathogen-related genes (MEKK1; PAD4; EDS1; PR1;WRKY33; bGS2) in uninfected transgenic overexpressing lines. Gene expression normalized tothe expression of the same gene detected inwild-type plants, Actin-2was used as reference gene. The comparative threshold cyclemethodwas used for determining differences betweentranscript copy numbers in wild-type and transgenic plants. Data represent average obtained for three independent transgenic lines ± standard deviation. Asterisks indicate significantdifferences between infiltrated and wild type plants, according to Student's t-test (n= 3; P b 0.05). (B) Determination of callose deposition.White arrows indicate one of multiple brightdots corresponding to callose deposition sites. Leaves from uninfected Arabidopsis transgenic and Col-0 plants were stained with aniline blue for callose visualization and photographedunder visible andUV light. The experimentwas repeated three timeswith similar results. Bars=200 μm. (C) Accumulation of H2O2 in uninfected transgenic andwild-type plants. Stainingof detached leaveswith 3,3′-diaminobenzidine revealed an accumulation ofH2O2 in leaves from Arabidopsis line overexpressingAtCYP19 gene. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)
8 G.V. Pogorelko et al. / Gene xxx (2014) xxx–xxx
ROS in non infected cells (Fig. 5) but lack of any conserved domains inAtCYP19 related to protein–protein interactions indicates that furtherin vitro pull-down assay or bio-molecular complementation experi-ments can provide experimental evidences of AtCYP19 interactors andlocate this immunophilin on a response pathway more precisely.
Our results reveal that AtCYP57 influences PAD4 expression,which isactivated downstream of MEKK1 (Fig. 5). EDS1 interacts with PAD4 toprovide basal immune response in plants and this complex is localizedboth in nucleus and cytoplasm (Zhu et al., 2011). Our data shows signif-icant increase of PAD4 expression in absence of EDS1 up-regulationtogether withWRKY33 induction and a subsequent enhanced transcrip-tion of PR1 and bGS2 (Fig. 3A) that strongly correlates with callose
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
accumulation in 35S:AtCYP57 plants (Fig. 3B). PR1 and PAD4 transcrip-tion is communicated via SA pathway (Chen et al., 1993; Zhang et al.,2013; Zhou et al., 1998) and are essential components of plant responseto pathogen invasion (Glazebrook et al., 1996, 1997; Louis et al., 2012;Wildermuth et al., 2001). WRKY33 is a member of large WRKYtranscription factors family in Arabidopsis (Eulgem et al., 2000) and rep-resents self-controlling pathogen-inducible transcription factor (Maoet al., 2011; Rushton and Somssich, 1998) involved in SA-dependentsignaling pathway (Yang et al., 1999). SA-dependent pathway wasshown to be activated by EDS1 in complex with PAD4 to performplant response to biotroph pathogens (Brodersen et al., 2006). Interest-ingly, our data shows that the overexpression of AtCYP57 can activate
rabidopsis thaliana immunophilin genes involved in the plant defense.1016/j.gene.2014.01.029
Fig. 4. The localization of C-GFP tagged AtCYP19-GFP (A), AtCYP57-GFP (B) and AtFKBP65-GFP (C) driven by its native promoters transiently expressed in N. benthamiana leaf epidermalcells after infection with P. syringae. A1, B1, C1 GFP fluorescence signal, A2, B2, C2 nuclear marker DAPI fluorescence signal, A3, B3, C3 merged GFP and DAPI fluorescence images. Bar =10 μm. A4, B4, C4 Western blot analysis of total proteins from corresponding infiltrated leaves.
9G.V. Pogorelko et al. / Gene xxx (2014) xxx–xxx
this pathway with only PAD4 activation (Fig. 3A) causing downstreamWRKY33 induction and as a consequence bGS2 expression which isrequired for callose production (Xie et al., 2011). The sub-cellular local-ization of AtCYP57 (Figs. 4B1–B3) indicates that this protein is presentin the cell nucleus and could participate directly in the regulation ofthese genes.
Constitutive expression of AtFKBP65 was shown to induce WRKY33and bGS2 gene transcription (Fig. 3A). Localization of AtFKBP65 in
Fig. 5. Proposed scheme of AtCYP19, AtCYP57, and AtFKBP65 interaction with plant cellpathogen-related patterns. AtCYP19 participates in reactive oxygen species (ROS) produc-tion inducingMEKK1 gene expressionwithout leucine-rich-repeat (LRR) and receptor-likekinase (RLK) mediated microbe/pathogen associated molecular patterns (PAMPs/MAMPs) signaling and activating mitogen-activated protein kinase (MAPK) cascade.AtCYP57 interacts directly with PAD4 and cause salicylic acid (SA) mediated activationof PR1, WRKY33, and bGS2. AtFKBP65 effects WRKY33 gene functioning inducing bGS2up-regulation.
Please cite this article as: Pogorelko, G.V., et al., Characterization of three Aresponse against Pseudomonas syringae, Gene (2014), http://dx.doi.org/10
nucleus (Figs. 4C1–C3) indicates that this protein can be a part of tran-scription regulation pathways upon pathogen infection (Fig. 5). Despitethe localization of AtFKBP65 exclusively to the cell nucleus, it does notcontain any nuclear localization signal (NLS). Previously it was shownthat AtFKBP65 is translocated to the nucleus of Arabidopsis cells duringthe recovery period after heat stress (Meiri et al., 2010). However, wedetected localization of the AtFKBP65-GFP fusion in the nucleus inN. benthamiana leaves that were not treated with high temperatures.In the case of yeast, it has been hypothesized that Cyclophilin A, whichalso lacks NLS, passes through nuclear pores passively due to its smallsize. However, AtFKBP65 was originally classified as a cytoplasmic pro-tein of more than 50 kDa (He et al., 2004) and cannot physically passinto the nucleus by diffusion (Mattaj and Englmeier, 1998; Talcott andMoore, 1999). Beside the FKBP conserved domain, the amino acidsequence of AtFKBP65 was found to contain tetratricopeptide andcalmodulin-bindingmotifs (He et al., 2004). Calmodulin-bindingmotifsare typical for proteins that regulate different processes by interactingwith a diverse group of cellular proteins (Harauz and Libich, 2009;Morad and Chau, 2004; Tan et al., 2011). The tetratricopeptide motifhas been identified in a wide variety of proteins, regulates the assemblyof protein oligomer complexes as well as protein–protein interactions,and is involved in numerous cellular processes including transcription,protein translocation, and degradation (Allan and Ratajczak, 2011;Blatch and Lassle, 1999; Smith, 2004). The presence of these protein–protein interaction motifs in AtFKBP65 leads us to hypothesize thatthis protein is delivered into the nucleus as a protein complex, similarto the WRKY33 complex, and acts as a modulator of the gene functionand protein expression during stress responses.
SinceWRKY transcriptional factor family is known to be highly mul-tifunctional proteins, our future experiments of genome wide tran-scriptome analysis together with proteome chip arrays may providemore insight into the cross-talk between the AtCYP57 and AtFKBP65signaling networks and their involvement in biotic stress responses.
Proposed pathways of induced plant tolerance to the pathogens donot exclude additional signaling pathways. We have shown the defectsof PTI in immunophilins knock-out lines which support our model
rabidopsis thaliana immunophilin genes involved in the plant defense.1016/j.gene.2014.01.029
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(Fig. 2C). However, it is known that immunophilins play a broad spec-trum of roles in stress tolerance. Indeed, they have been associatedwith transcriptional regulation of stress related genes, and are knownto be involved in Na+ or Cl- ions traffic control (Arévalo-Rodríguezand Heitman, 2005; Sekhar et al., 2010). Moreover, the function ofthese proteins in cell cycle regulation may collaterally converge withresistance to pathogens by cellular dedifferentiation (Grafi et al., 2011).
We have shown that AtCYP19 can activate ROS-mediatedMAPK cas-cade without the presence of invaders' effectors and both AtCYP57 andAtFKBP65 provide callose deposition in the cell wall preventing fastpenetration of P. syringae into plant tissues by SA dependent and inde-pendent pathways respectively. However, we do not exclude the possi-bility that selected immunophilins can also trigger Effector-TriggeredImmunity (ETI) (Fig. 5) by direct recognition of P. syringae effectorswhich can be analyzed in future work by yeast two hybrid system orrelated techniques. Histochemical analysis of the promoter-drivenGUS gene allowed us to show activation of ectopic expression ofeach of the AtCYP19, AtCYP57 and AtFKBP65 genes in the presence ofP. syringae with the lack of signal transduction from invaded tissues tosurrounding areas improving that all three immunophilins are involvedin local stress response. Since PAD4 was previously reported to act as alocal defense (Louis et al., 2012) and we detected local induction ofAtCYP57, we propose AtCYP57 as a possible interactor with PAD4 andour future experiments will help us to prove or refuse this suggestion.Overall characterized three genes of plant immunophilin family ispromising tool for future investigations in the area of plant-pathogeninteractions and innate immune system.
Conflict of interest
None.
Acknowledgments
Authors thank Andrew Foundree, a research scientist at PioneerCompany (Johnston, IA), and William Rutter (Department of PlantPathology, Iowa State University) for reading and discussing this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2014.01.029.
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