in silico study of interaction between rice proteins enhanced disease susceptibility 1 and...

In silico study of interaction between rice proteins enhanced diseasesusceptibility 1 and phytoalexin deficient 4, the regulators of salicylic

acid signalling pathway

INDRA SINGH1 and KAVITA SHAH2,*1Department of Bioinformatics, MMV, 2Institute of Environment & Sustainable Development,

Banaras Hindu University, Varanasi 221 005, India

*Corresponding author (Fax, +91-542-2307225; Email, [email protected])

Enhanced disease susceptibility 1 (EDS1), a plant-specific protein has homology with the eukaryotic lipase in theirN-terminal halves and a unique domain at its C-termini. EDS1 is known to be an important regulator of biotic stressand an essential component of basal immunity. EDS1 interacts with its positive co-regulator phytoalexin deficient4 (PAD4), resulting in mobilization of the salicylic acid defence pathway. Limited information regarding thisinteraction in rice is available. To study this interaction, a model of EDS1 and PAD4 proteins from rice was generatedand validated with Accelrys DS software version 3.1 using bioinformatics interface. The in silico docking between thetwo proteins showed a significant protein–protein interaction between rice EDS1 and PAD4, suggesting that they forma dimeric protein complex, which, similar to that in Arabidopsis, is perhaps also important for triggering the salicylicacid signalling pathway in plants.

[Singh I and Shah K 2012 In silico study of interaction between rice proteins enhanced disease susceptibility 1 and phytoalexin deficient 4, theregulators of salicylic acid signalling pathway. J. Biosci. 37 563–571] DOI 10.1007/s12038-012-9208-4

1. Introduction

Salicylates (SA) are phenolic compounds known to possessmedicinal properties (Rainsford 2004). SA is also well estab-lished as a signalling molecule in plant immune response(Volt et al. 2009; Chuanfu and Mou 2011). In order tosurvive, plants need to respond to numerous environmentalstresses and reprogram their metabolism and growth accord-ingly (Rietz et al. 2011). Rice plants respond to differentstressors via a number of mechanisms (Shah and Nahakpam2011). Availability of rice (Oryza sativa) genome sequences,information generated from genomics and proteomics stud-ies and in silico computational bioinformatics tools, all set anew platform for the management of environmental stressesin rice. The central convergence point in stress signallingunder different stresses is the formation of reactive oxygenintermediates (ROI), which, in turn, modulate various sig-

The EDS1 protein, encoded by the enhanced susceptibilitygene1 (EDS1) along with its coregulator phytoalexin defi-cient4 gene (PAD4), is important in the regulation of theplant defence pathway involving SA signalling (Weirmer et al.2005). EDS1 and PAD4 are required for the SA accumula-tion and for the defence pathway involving ROI-derivedsignals in plant cells (Kotchoni and Gachomo 2006) result-ing from abiotic, xenobiotic or biotic environmental stresses(Singh et al. 2012), and is shown in figure 1. Preliminarystudies suggest that EDS1 protein together with PAD4 formsa regulatory node that serves as a stimulus for environmentalstresses (Rietz et al. 2011). It is documented that the expres-sion of EDS1 and PAD4 are induced by salicylic acid (Singhand Shah 2012). A direct interaction of EDS1 with PAD4 isnecessary for basal resistance and is related to the transcrip-tional up-regulation of PAD4 and immobilization of thesalicylic acid defence pathway in Arabidopsis (Rietz et al.

Published online: 25 June 2012

nalling pathways including the SA signalling pathway inplants (Kotchoni and Gachomo 2006).

http://www.ias.ac.in/jbiosci J. Biosci. 37(3), July 2012, 563–571, * Indian Academy of Sciences 563

Keywords. Enhanced disease susceptibility 1; in silico; phytoalexin deficient 4; protein–protein interaction; salicylate

2011). Dynamic changes in the EDS1–PAD4 complex arevery important for coordination of signalling between nucle-

ar and cytoplasmic compartments, which is needed for com-plete immune response against stress in turnip (Zhu et al.2011). SA and its derivatives are known to stimulate EDS1and PAD4 expression as a part of the positive feedback loopthat amplifies resistance locally and systemically (Feys et al.2001; Volt et al. 2009). Proteomic studies in Arabidopsisthaliana revealed that the EDS1 pathway components in-clude two putative lipases that are positive regulators, PAD4and senescence-associated gene101 (SAG101) (Feys et al.2005). EDS1 interacts with PAD4 and SAG101 at differentsubcellular locations including nucleo-cytoplasmic EDS1-PAD4 and nuclear EDS1-SAG101 heterodimers (Feys etal. 2005). Prominent among the EDS1-dependent up-regulated genes are components of SA biosynthesis andsignalling [Isochorismate synthase1 (ICS1), Pph susceptible3 (PBS3) and CaM binding protein (CBP60g)] and flavin-dependent mono-oxigenase 1(FMO1), which positivelyregulates the SA-independent branch of EDS1 pathway(Wildermuth et al. 2001; Bartsch et al. 2006; Mishina andZeier 2006; Orkent et al. 2009; Wang et al. 2009) andpathogenesis related 1 (PR1), a commonly used SA responsemarker (Laird et al. 2004). Therefore, a key step of EDS1nuclear action is to stimulate the SA pathway. Although thepresence of both nuclear and cytosolic EDS1 regulator iswell documented, their roles are still unclear.

Earlier we reported a large number of hypothetical pro-teins encoded by genes of the SA-JA pathway in rice that areyet to be modelled for structure and protein–protein interac-tion studies so as to assign proper function to them (Singhet al. 2012). The present work uses bioinformatics tools forhomology modelling of EDS1 and PAD4 proteins from ricein silico. Interaction of rice EDS1 with its signalling partner

PAD4, in order to better understand how EDS1 coordinatesresponses to multiple stress stimuli, forms an important partof this work.

2. Methods

2.1 Retrieval of the sequences

Amino acid sequences of EDS1 and PAD4 of Arabidopsiswere retrieved at NCBI (http://www.ncbi.nlm.nih.gov).Orthologous sequences that were un-annotated hypotheticalproteins at the time of work and corresponded to the proteinsin rice were obtained through BLASTp program (Altschul etal. 1990). Arabidopsis EDS1 (accession no. AAD20950.1)protein is 623 amino acids in length and PAD4 (accessionno. NP_190811.1) protein is 541 amino acids long.

2.2 Template identification and homology modelling

The three-dimensional model of EDS1 and PAD4 proteins ofrice were constructed by homology modelling. BLAST pro-gram against PDB (Bernstein et al. 1977) was used to searchand identify template proteins for rice EDS1 and PAD4proteins. Template selection was carried out with a cut-offscore of >32% sequence identity and >50% sequence posi-tivity. Crystal structure of Lipase Class II proteins with PDBID: 1DT3 and 1DT5 were chosen as the template for mod-elling EDS1 and PAD4 proteins, respectively. Model build-ing was performed using the program Accelrys DS (http://accelrys.com/products/discovery-studio). Several models at

Figure 1. Involvement of EDS1 regulatory node and its coregulators PAD4 and SAG101 in abiotic and biotic stress responses in plants,affecting the SA signalling pathway. ACD11: Accelerated Cell Death 11 gene; LSD1: Lesions Stimulating Disease 1 gene; EDS1: EnhancedDisease Susceptibility 1; SAG101: Senescence Associated gene 101; PAD4: Phytoalexin Deficient 4 gene; MAPK4: mitogen activatingprotein kinase 4; HR: hypersensitive reaction; SA: salicylic acid; ROI: reactive oxygen intermideates; ICS1: Isochorismate Synthase1;PBS3: Pph susceptible 3; CBP60g: CaM binding protein; PR1: Pathogenesis related 1.

564 Indra Singh and Kavita Shah

J. Biosci. 37(3), July 2012

http://www.ncbi.nlm.nih.gov

http://accelrys.com/products/discovery-studio

various refinement levels and library schedules wereobtained

2.3 Model optimization and evaluation

The several models obtained above were subjected to energyminimization using the steepest descent technique by in

vaccuo computation with GROMOS96 43B1 parameter set,through Swiss-Pdb Viewer. All models were validated usingthe program PROCHECK (http://nihserver.mbi.ucla.edu/SAVES_3/saves.php). The backbone conformation of themodel was inspected by the Ramachandran plot at PRO-CHECK server. The best model had a Procheck score of−0.19 for EDS1 and −0.20 for PAD4.

Figure 2. BLASTp results showing conserved domains (red boxes) in rice (Sbjct) with Arabidopsis (Query) EDS1 protein andnucleophilic elbow (GHSSG), and catalytic triads SSG and DII (blue boxes). Green box shows the conserved non-polar leucine residueinvolved in flexibility of binding of EDS1 to PAD4.

In silico study of interaction between EDS1 and PAD4 565


http://nihserver.mbi.ucla.edu/SAVES_3/saves.php

2.4 Protein–protein interaction and analysis

EDS1–PAD4 complex formation and protein–protein interac-tion between EDS1 and PAD4 were studied using HEX6.3software (http://hex.loria.fr/dist/index.php). Analysis ofprotein–protein interaction was carried out in Accelrys DSviewer. HEX6.3 parameters used in the protein−proteininteraction included correlation type: shape and electrosta-tistics; grid dimension: 0.6; receptor range (EDS1): 45;

ligand range (PAD4): 45; distance range: 40; receptor stepsize: 5.5 and twist scan size: 5.5.

3. Results

3.1 Retrieval of the protein sequences and analysis

Amino acid sequences of EDS1 and PAD4 of rice obtainedat NCBI using the Arabidopsis 623-amino-acid-long EDS1

Figure 3. BLAST results showing conserved domains in rice (Sbjct) With Arabidopsis (Query) PAD4 protein and nucleophilic elbow(GHSSG), and catalytic triads SSG and DII (blue boxes).



http://hex.loria.fr/dist/index.php

(accession no. AAD20950.1,) protein and the 541-amino-acid-long PAD4 (accession no. NP_190811.1) proteinresulted in rice protein orthologues of 621-amino-acid-longEDS1 (accession no. EAZ08987.1) and 664-amino-acid-long PAD4 (accession no. OsJ_33269) proteins (figure 2).Rice EDS1 contains three catalytic triads, two SSG and oneDII type; however, in the Arabidopsis EDS1 protein onlyone SSG catalytic triad was present (figure 2). Rice proteinEDS1 has a nucleophilic elbow ‘GHSSG’ at position 141–145 and two lipase-specific catalytic triads 143SSG145 and207DII209 (figure 2). In addition, one more SSG triad inEDS1 protein was noted at position 37SSG39. The 37SSG39

and 207DII209 triads, although present in rice and reported atNCBI to have lipase like function, were not seen to partic-ipate in binding with PAD4, indicating that these two cata-lytic triads on rice–EDS1 might be responsible fortriacylglycerol lipase-like activity alone and therefore mightnot be involved actively in binding to PAD4. Sequenceanalysis revealed high conservation of the non-polar aminoacids in the conserved domain 262ELSPYRP269 of Arabidop-sis which corresponded to position 284–291 in rice EDS1protein sequence (figure 2). Conserved domains, nucleophil-ic elbow and catalytic triad in PAD4 protein are shown asfigure 3. In Arabidopsis, the PAD4 protein contains only oneSSG catalytic triad, but in rice PAD4 two catalytic triads(one SSG and one DII type) were seen. This suggests thatEDS1 and PAD4 proteins in rice vary from that of Arabi-dopsis, conferring additional functions that need to be

ascertained through wet lab studies. Sequence analysis withSignal IP 4 server (http://www.cbs.dtu.dk/services/SignalP)revealed the presence of a signal peptide and a cleavage sitebetween position 26 and 27 on rice PAD4; however, nosignal peptide was obtained on rice EDS1 (figure 4).

3.2 Template identification and homology modelling

BLAST program against PDB resulted in solved crystalstructure of Lipase Class II proteins as best templates PDBID: 1DT3 and 1DT5 for rice proteins EDS1 and PAD4,respectively. The modelled structures of EDS1 and PAD4

Figure 4. PAD4 protein of rice showing cleavage site between positions 26 and 27 as obtained by Signal IP-4.8.

Figure 5. The three-dimensional structure of EDS1 protein homo-dimer of rice as modelled by Discovery Studio v 3.1. using PDBID: 1DT3 as template and accession no. EAZ08987.1 as target,showing helix (red), B-sheet (blue) and loops (white).



http://www.cbs.dtu.dk/services/SignalP

are shown in figures 5 and 6, respectively. In silico study ofmodelled EDS1 structure forms a homodimer (figure 5).

3.3 Model optimization and evaluation

The EDS1 and PAD4 protein models obtained initially wereassociated with −9335.431 kJ and −18581.992 kJ energy respec-tively, which upon energy minimization by the steepest descenttechnique reduced to −22907.027 kJ and −28248.201 kJ, respec-tively. Ramachandran plot analysis showed <2.5% residues indisallowed regions for both the proteins, indicating that themodels were of good quality (figure 7).

3.4 Protein–protein interaction and analysis

Based upon the lowest energy associated with the EDS1 andPAD4 protein models of rice, the best models were selected forfurther interactive studies. EDS1–PAD4 complex formation andprotein–protein interaction between EDS1 and PAD4were stud-ied using HEX6.3 software (figure 8). Analysis of protein–protein interaction was carried out in Accelrys DS viewerand both EDS1 and PAD4 shared four conserved triacylgly-cerol lipase-specific motifs: EKRIVFTGHSSGGSIA,HPFCVTFGAPLVGDNL, LGTLTSFIELSPY and LYRRL-VEPLDIANYYRHSKNEDTGSYLSKGRPRRY present atpositions 135–150, 170–184, 277–289 and 490–524 onEDS1 and 134–157, 170–184, 267–295 amd 491–532 onPAD4 proteins, confirming these two proteins to have func-tions similar to lipases in rice plants . Rice PAD4 interactedwith the whole length protein of rice EDS1. The region 73–

Figure 6. Model of three-dimensional structure of PAD4 proteinas modelled by Discovery Studio v 3.1. using PDB ID: 1DT5 astemplate and accession no. OsJ_33269 as target, showing helix(red), B371 sheet (blue) and loops (white).

Figure 7. Ramachandran plot for EDS1 and PAD4 proteins of rice showing >82% allowed regions in the two proteins.

Figure 8. EDS1-PAD4 complex; the blue circle denotes interac-tion between two proteins.



130 of EDS1 protein is a large domain that interacts with twodifferent domains 426LNCASLATRLGRITPCRAQIEW447

and 420AHFYQLLVEPL430 of PAD4. Protein domain448DANTGYYDA456 in the helix of PAD4 interacts with369RQMSSTIVGGLELS382 of EDS1 protein. Domain216HVVSQHDVVPRLLFCPLNVIPVH238 of PAD4 inter-acts with a large domain of EDS1 at position 155–185. Apartfrom helix–helix and helix–loop interactions there appearβ-sheet–helix and β-sheet–loop interactions as well as thosebetween the EDS1 and PAD4 proteins of rice (figure 9).

4. Discussion

EDS1 and PAD4 proteins are required for salicylate-mediated defence potentiation together with ROI-derivedsignals in plants, and EDS1 acts as a scaffold for the PAD4activities. Coordination between the EDS1 cytoplasmic andnuclear pool via the nuclear pore trafficking is needed fordefence regulation (Cheng et al. 2009; Garcia et al. 2010).

Sequence analysis revealed high conservation of the non-polar amino acid leucine at position 255–265 among mono-and dicotyledonous plant species (including rice) that issuggested to contribute in conformational flexibility betweendifferent states of EDS1 to allow PAD4 binding (Wiermeret al. 2005). Rice protein EDS1 had a nucleophilic elbow‘GHSSG’ at position 141–145 and two catalytic triads143SSG145 and 207DII209 (figure 2) which are characteristicof lipases in general, but involvement of 143SSG145 alonesuggests that SSG at position 143 is primarily involved inprotein–protein interactions involving EDS1. The two cata-lytic triads (SSG and DII) seen on EDS1 forming an activesite pocket is perhaps for interaction with the substratetriacylglycerol, and therefore these triads do not interactdirectly with PAD4 protein in rice as observed in our study.Orthologous sequence for EDS1 and PAD4 of rice retrievedfrom NCBI using BLAST server resulted in the 621-amino-acid-long EDS1 protein and 662-amino-acid long PAD4protein which was significantly longer proteins when com-pared to Arabidopsis. EDS1 is present in cytoplasm as

Figure 9. Interaction between amino acid residues in different domains of EDS1 (red)–PAD4 (blue) complex.



suggested by the absence of a signal peptide in EDS-1,whereas PAD4 has a signal peptide allowing it to move tonucleus as well. EDS1 and PAD4 interact forming a proteincomplex involved in the regulation of the salicylic aciddefence pathway. There are four triacylglycerol lipase moi-eties, suggesting that EDS1 and PAD4 proteins from ricehave lipolytic function.

EDS1 is capable of nuclear transport receptor-mediatedshuttling between the cytoplasm and nucleus. Garcia et al.(2010) showed that in transgenic Arabidopsis by enhancingEDS1 export from inside nuclei via attachment with addi-tional nuclear export sequence or coordinated release ofEDS1 to the nucleus by glucocorticoid receptor, it is theEDS1 nuclear pool that is essential for resistance and fortranscriptional reprogramming. It is advocated that cytosolicEDS1 activity promotes cell death in response to oxidativestress signals emanating from the chloroplasts (Mateo et al.2004; Muhlenbock et al. 2008; Straus et al. 2010), and this iscounterbalanced by transcriptional reprogramming in thenucleus in order to moderate potentially destructive cellularevents (Garcia et al. 2010). EDS1 is also required for resis-tance conditioned by nucleotide-binding leucine-rich repeat(NB-LLR) immune receptors that have the terminal Toll/Interleukin-1 Receptor (TIR) domain (Aarts et al. 1998;Jones and Dangl 2006; Venugopal et al. 2009). EDS1 isreported to signal after TIR-NB-LRR immune receptor acti-vation and upstream of the transcriptional reprogramming ofdefence genes, production of resistance hormone salicylicacid and host cell death (Feys et al. 2001; Wirthmueller et al.2007). Similar to the biochemical analysis reports of EDS1–PAD4 complex in E. coli (Macarthur and Thornton 1991),the existence of four different binding domains were alsoobserved in this study on rice (figure 9). This provides a highprobability of formation of several transient interactionsbetween EDS1 and PAD4, including helix–helix andhelix–loop interactions, and β-sheet–helix and β-sheet–loopinteractions between the two proteins involving differentdomains on EDS1 and PAD4 that might play a significantrole in EDS1–PAD4 complex formation and stability. Thiswould also mean that there is a weak association with EDS1and PAD4 complex formation in rice. PAD4 is reported tobe similar to a ferulic acid esterase from Aspergillus niger,and therefore it is also possible that its substrate is not a lipid(Jirage et al. 1999). There have been very limited studies onrice plant EDS1 and PAD4 genes and proteins, and thereforemore wet lab and in silico analyses are needed to validate theinvolvement of these two proteins in SA signalling in rice.Recently, formation of EDS1–PAD4–SAG101 ternary com-plex has been reported in turnip (Zhu et al. 2011), but not inE. Coli (Rietz et al. 2011).

The results, therefore, suggest that EDS1 naturally exists asa homodimer in rice. It forms an oligomeric complex withPAD4 (as no other protein like SAG101 is present). A single

EDS1 domain does not form a complex with PAD4. Thereappears to be the possibility of weak interaction (van derWaalsand hydrogen bonds) between EDS1 and PAD4 involving twoor more different domains on PAD4 in rice. At present, a studyto check if EDS1 associates with SAG101 together with PAD4forming a ternary complex in rice is being carried out and thiswill be helpful in explaining the cooperation between EDS1and PAD4/SAG101 proteins. Also, it will be interesting tostudy the complexation of EDS1–SAG101 and if EDS1 canbridge PAD4 and SAG101 in rice.

Acknowledgements

IS gratefully acknowledges the Department of Science andTechnology, New Delhi, for providing financial supportunder the DST-Women Scientist DST Grant No. 100/IFD/5462/2010–11. Financial support by University Grants Com-mission, New Delhi, grant No. 39–231/2010 to KS, is alsoacknowledged. We are thankful to the School of Biotechnol-ogy, BHU, for technical support.Both authors have contributed equally to this work and

there is no conflict of interest.

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