International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(8), 20-26, August (2013) Int. Res. J. Biological Sci. International Science Congress Association 20 Identification of Rice Kinesin 13A Interacting Proteins through Yeast Two-Hybrid ScreeningYuyun Sugiharti and Mukhamad Su’udi1,2*Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, KOREA National Academy of Agricultural Science, RDA, Suwon 441-707, KOREAAvailable online at: www.isca.in Received 28th April 2013, revised 10th May 2013, accepted 15th June 2013Abstract By using full-length rice kinesin 13A protein (OsKIN13A) as a bait construct and rice cDNA library as a prey, we obtained several interacting proteins through yeast two-hybrid (Y2H) screening. These interacting proteins were selected by beta-galactose filter assay after growing on SD-TLH media containing 25 mM of 3-aminotriazole (3-AT). All of selected interacting proteins were confirmed by sequencing and annotated according to the rice genome database, and grouped into seven functional categories, i.e. cellular processes, transport and binding protein, metabolic activity, signal transduction, molecular chaperone and other category. This study suggests that OsKIN13A protein is associated with wide range of biological processes such as development, signaling and immunity. Keywords: OsKIN13A, Y2H, binding domain, cDNA library Introduction Kinesins have been known as family of motor proteins that move along microtubules. Initially, kinesin was identified in squid, and has been reported as a microtubule-based protein with high motility1,2. Afterwards, plant kinesins were also discovered in tobacco organs such as pollen tubes and phragmoplasts3-5. The members of kinesins family have unique features, i.e., the existence of conserved motor domain with ATP- and MT-binding sites. However, this family has relatively less- and non-conserved coiled-coil region and tail domain, respectively.Comprehensive genomic study has classified kinesins into 14 subfamilies, according to the conserved motor domain. In the genome of Arabidopsis thaliana, at least 61 genes have been identified to encode microtubule-based motor kinesins. Some of them have been characterized physiological- and genetically using mutant lines. For example, fragile mutant (FRA1), an Arabidopsis kinesin-4, is involved in oriented deposition of cellulose microfibrils in fiber cell walls. Kinesin 12A and 12B play prominent roles in cytokinesis during male gametogenesis. Likewise, Arabidopsis kinesin-14 is indispensable for early spindle formation10. Recently, kinesin-like protein 1 (KP1) has been reported to interact with voltage-dependent anion channel 3 (VDAC3), a mitochondrial outer membrane protein11. In contrast to Arabidopsis, rice genome evolved less number of kinesins. Current study reported that the rice cultivar (cv.) japonica contain 41 kinesins, while in cv. indica has larger number (45 kinesins)12. Functional studies in rice were also reported within the recent years. For example, Pollen Semi-Sterility1 (PSS1) encodes a kinesin-1-like protein, is crucial for male gametogenesis and anther dehiscence13. Brittle Culm12BC12), a gene encoding a rice kinesin-4 protein, is involved in maintaining plant mechanical strength through regulation of cell wall composition14. BC12 also play important role in cell elongation by regulating GA biosynthetic pathways15. In addition, BC12 has been found to interacts with CDKA;3, a cell cycle protein, through yeast two hybrid analysis. The yeast two-hybrid system (Y2H) is one of the most powerful and versatile methods to study protein-protein interaction as well as characterizing a protein’s functions. This method is able to identify novel proteins interact with the bait construct through the recovery of yeast GAL4 transcription16. Using this strategy, we aimed to identify proteins that interact with rice kinesin 13A (OsKIN13A). In spite of its critical function in maintaining microtubule-related processes, kinesins have been reported to play role in many other biological processes. Functional studies in plant revealed that kinesins are indispensable for plant architecture and development. Although research about kinesins has been started since the last decade, the investigation of its member is still plenteous due to the huge number of this family in the plant. In this study, we characterize the rice kinesin 13A (OsKIN13A) in the respect of protein-protein interaction. Material and Methods Sequence Analysis: The nucleotide and amino acid sequences of OsKIN13A were retrieved from the rice genome database (http://rice.plantbiology.msu.edu/). Alignment between cDNA and genomic DNA was observed through Spidey program (http://www.ncbi.nlm.nih.gov/spidey/index.html). Molecular International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(8), 20-26, August (2013) Int. Res. J. Biological Sci. International Science Congress Association 21 weight (MW) and isoelectric point (pI) was calculated using the prediction site: http://web.expasy.org/compute_pi/. In addition, protein domain feature was determined with Prosite (http://prosite.expasy.org/cgi-bin/prosite/). A phylogenetic tree was constructed as mentioned previously17,18. RNA Isolation and RT-PCR Analysis: Total RNA was isolated from leaves, roots, flower, and seeds using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). First-strand cDNA was synthesized at 37C for 1 h in a total reaction volume of 25 l containing 2 g of total RNA, 1.5 g oligo (dT)18primer, 2.5 mM dNTP and 200 units of M-MLV reverse transcriptase (Promega, Madison, WI, USA). For studying the expression of OsKIN13A, 1.0 l cDNA sample from the RT reaction was used for PCR in 25 l total reaction mix containing 10 picomole of OsKIN13A-RTF (5-AAC TCT AGG ACG GTG ATG ATC TCT TG-3) and OsKIN13A-RTR (5-GTT GCT GTT GCA ACT GCT TTG CCT G-3) primers. The PCR mixture was initially denatured at 95C for 5 min and then subjected to 27 cycles of the following conditions: 95C for 15 s, 58C for 15 s, 72C for 1 min with a final extension at 72C for 5 min. Amplification of rice Actin was used to validate equal amounts of cDNA per reaction. Primers for Actin were: ActinRTF (5-AAC TGG TAT GGT CAA GGC TGG GTT-3) and Actin-RTR (5-GCA ATC CAC ATC TGC TGG AAT GTG C-). PCR conditions were: 95C for 5 min, followed by 23 cycles of 95C for 15 s, 58C for 15 s, and 72C for 1 min, with a final extension at 72C for 5 min. Detection of PCR product was carried as mentioned previously19-21. Yeast Two-hybrid Assay: For bait vector construction, the ORF of OsKIN13A was PCR-amplified using Pfu DNA polymerase with the following primers: 5-GAT GGG GGA CTC CGG GGA CGC CGT CAT-3 and 5-GGA TCC TTA TCT GGA AGA TTT CTT ACG GCT G-3 (the start codon and BamHI site is underlined). The pGBT9 vector was sequentially digested with SmaI and BamHI. Subsequently, the PCR-amplified product of OsKIN13A was digested with BamHI and ligated into pGBT9 vector to create pGBT9-OsKIN13A. The cDNA library (prey) was made from rice according to the HybriZAP two-hybrid cDNA Gigapack cloning kit manual (Stratagene, La Jolla, CA, USA). Total cDNA of the phagemid form was obtained by the mass in vivo excision method. The yeast (Saccharomyces cereviseae) strain YRG-2 (genotype: MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS::UASGAL1-TATAGAL1-HIS3, URA3::UASGAL417mers(x3)TATACYC1-lacZ) was transformed with the binding domain pGBT9-OsKIN13A. The yeast cells containing the binding domain were co-transformed with 100 g of the HybriZAP cDNA library plasmid DNA and salmon sperm carrier DNA by the lithium acetate method22. Transformants were selected on the synthetic dropout medium lacking tryptophan, leucine, and histidine (SD-TLH). The transformants, which appeared after 3-5 days incubation at 30C, were then grown on an SD-TLH plate containing 5 mM and then 25 mM 3-aminotriazole (3-AT). Yeast colonies which still survived on media containing 25 mM 3-AT were tested for the beta-galactosidase activity by the filter assay23. The colonies that turned blue in less than 6 h were collected and used as the template in colony PCR reaction as described previously24. The primers for amplification were GAL4 AD-forward and -reverse with following PCR procedure: the denaturation condition was 7 min at 95C, followed by the amplification reaction for 1 min at 95C, 1 min at 55C, and 3 min at 72C for 35 cycles, prior to a final extension for 7 min at 72C and storage at 4C. The PCR product was then purified with a QIAEX II kit (Qiagen, Valencia, CA, USA) and confirmed by DNA sequencing. Nucleotide sequences obtained from DNA sequencing were used for gene annotation by BLAST against NCBI (http://www.ncbi.nlm.nih.gov) and rice genome database (http://rice.plantbiology.msu.edu/).Results and Discussion Expression Pattern, Molecular Structure and Sequence Analysis of OsKIN13A The expression pattern of OsKIN13Awas investigated through RT-PCR with gene-spesific primers, based on the steady-state mRNA levels in different rice tissues and /or organs. The results showed that this gene is indeed expressed in all tissues /or organ examined (figure-1). OsKIN13A gene transcript was highly expressed in flower and seed. The expression level was lower in leaf and root, respectively. The OsKIN13A is most likely expressed constitutively through the rice life cycle. This result indicates that OsKIN13A might play potential role during vegetative and reproductive development. The molecular structure of OsKIN13A is shown in figure-2A. OsKIN13A consists of nineteen exons and eighteen introns. The OsKIN13A contained an open reading frame (ORF) of 3,300 bp encoding a polypeptide of 1,099 amino acid residues with a calculated molecular mass of 122.22 kDa and pI of 6.14. Kinesin motor domain was determined to be located at amino acid between 188 and 458 (figure-2B). More specifically, a short motif (GKISFIDLAGSE) located at amino acid number 426 to 437 was predicted as a core motor domain signature. Glycine-rich region was found in amino acid residue 27-50. In addition, nuclear localization signal (NLS) was determined at amino acid residue 965-979. Database search revealed that OsKIN13A is located at chromosome number 5 (LOC_Os05g06280). Phylogenetic tree was generated by comparing OsKIN13A protein with other previously reported kinesins in rice. As shown in figure-2C, OsKIN13A has high identity with LOC_Os01g43580. Screening Proteins that Interact with OsKIN13A: In general, proteins interact with other biomolecules to execute their functions. With yeast-two hybrid (Y2H) system, proteins that interact with OsKIN13A were determined and classified International Research Journal of Biological Sciences ________________________________________________ Vol. 2(8), 20-26, August (2013) International Science Congress Association according to their function. These proteins may functions as co activator, inhibitor or substrate. The Y2H was conducted to identify proteins that interact, as partner or target proteins, with OsKIN1 transfromants were grown under SD- Trp plate. To reduced the amount of transformants, screening was conducted by transferred about 5 SD- TLH containing 5 mM and 25 mM of 3 AT). Approximately, more than 100 transformants were able to grow under media with 25 mM 3-AT. The transformants were also tested for LacZ activation. Finally interacting proteins (table-1). To make better understand, candidate proteins were classified into several groups due to their functional role in biological processes ( These proteins were grouped into six functional cellular processes (CP), transport and binding protein (TB), metabolic activity (MA), signal transduction (ST), molecular chaperone (MC), and other category. The example of proteins that is involved in cellular process es are elongation factor, expansin, tubulin an d membrane protein. Cyclin kinase C was also included in this category. The example of proteins that is involved in transport and binding are transferase, reductase, and zinc finger protein. Metabolic activity group consist of several functional proteins such as cellulose, carboxylase, dehydrogenase, and synthase. Proteins such as PGIP2, B12D, and Myb transcription factor were grouped into signal transduction category. Three heat shock protein family (DnaJ, Hsc70, Hsp81) and an immunophilin were c into molecular chaperone. Of seven categories, proteins involved in transport/ binding are mostly interacts with OsKIN13A. Interestingly, four proteins appeared twice during screening process. These are (C3HC4-type RING finger) prot ein, heat shock cognate 70 kDa protein, senescence- associated protein (B12D) and ferredoxin thioredoxin reductase. Hence, OsKIN13A likely has notable Expression pattern of the International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association according to their function. These proteins may functions as co - to identify proteins that interact, as partner or target proteins, with OsKIN1 3A. More than 1000 Trp -Leu-His (SD-TLH) plate. To reduced the amount of transformants, the second by transferred about 5 00 colonies into TLH containing 5 mM and 25 mM of 3 -aminotriazole (3- transformants were able to The transformants were activation. Finally , we observed 81 To make better understand, all into several groups due to their functional role in biological processes ( table-1, figure-3). functional categories, i.e. processes (CP), transport and binding protein (TB), metabolic activity (MA), signal transduction (ST), molecular chaperone (MC), and other category. The example of proteins es are elongation factor, d membrane protein. Cyclin -dependent kinase C was also included in this category. The example of proteins that is involved in transport and binding are transferase, reductase, and zinc finger protein. Metabolic activity group proteins such as cellulose, carboxylase, dehydrogenase, and synthase. Proteins such as PGIP2, B12D, and Myb transcription factor were grouped into signal transduction category. Three heat shock protein family (DnaJ, Hsc70, Hsp81) and an immunophilin were c ategorized Of seven categories, proteins involved in transport/ binding are mostly interacts with OsKIN13A. Interestingly, four proteins appeared twice during screening process. These are zinc finger ein, heat shock cognate 70 kDa associated protein (B12D) and ferredoxin thioredoxin reductase. Hence, OsKIN13A likely has notable interaction with these proteins. Zing fingers are protein that can adapt one or more zinc ions for their addition, most of zing finger proteins in plant are transcription factors and involved in several numbers of biological processes such as interactions with other molecules and stress regulation25,26 . More specifically, proteins play a key role in ubiquitination pathway shock cognate 70 kDa protein (Hsc70) is a member of heat shock protein 70 family that can binds nascent polypeptide to facilitate correct protein folding proteins, Hsc70 also has ATPase activity Plant senescence related with hormonal signaling and maturation or aging. In addition, senescence can be induced by environmental stresses or pathogen infection associated with programmed cell death (PCD senescence- associated protein (B12D) interacts with OsKIN13A. B12D has been reported to be accumulated during seed maturation and leaf senescence reductase is potent candidate that interacts with O This enzyme involved in photosynthetic action, especially during light- dependent reaction i.e. coverts ferredoxin into thiol group. Another interesting finding is OsKIN13A interacts with Cyclin dependent kinase C (CDKC). CDKC is orthologues of C human protein that critical for embryo development. In Arabidopsis , CDKC and its partners (CYCT1) regulate immunity responses against responsible for normal growth morphology, trichome development, and flowering time interacts with polygalacturonase inhibiting protein 2 (PGIP2). PGIP have been reported to restrain fungal and bacterial invasion in Arabidopsis , tomato, grapevine, tobacco and cabbage36-40 . Interestingly, of five to six PGIP me is the most powerful molecule to inhibit polygalacturonase activity secreted by fungal pathogens Figure-1 Expression pattern of the OsKIN13A OsActin was included as an internal control International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 22 interaction with these proteins. Zing fingers are protein that can adapt one or more zinc ions for their folding stabilization. In addition, most of zing finger proteins in plant are transcription factors and involved in several numbers of biological processes such as interactions with other molecules and stress -tolerance . More specifically, C3HC4-type RING finger proteins play a key role in ubiquitination pathway 27,28. Heat shock cognate 70 kDa protein (Hsc70) is a member of heat shock protein 70 family that can binds nascent polypeptide to facilitate correct protein folding 29. Like other heat shock proteins, Hsc70 also has ATPase activity 30. Plant senescence related with hormonal signaling and maturation or aging. In addition, senescence can be induced by environmental stresses or pathogen infection associated with programmed cell death (PCD 31,32. Our result identified associated protein (B12D) interacts with OsKIN13A. B12D has been reported to be accumulated during seed maturation and leaf senescence 33,34. Ferredoxin thioredoxin reductase is potent candidate that interacts with O sKIN13A. This enzyme involved in photosynthetic action, especially dependent reaction i.e. coverts ferredoxin into thiol Another interesting finding is OsKIN13A interacts with Cyclin - dependent kinase C (CDKC). CDKC is orthologues of C dk9, a human protein that critical for embryo development. In , CDKC and its partners (CYCT1) regulate immunity responses against Cauliflower mosaic virus (CaMV), responsible for normal growth morphology, trichome development, and flowering time regulation35. OsKIN13A also interacts with polygalacturonase inhibiting protein 2 (PGIP2). PGIP have been reported to restrain fungal and bacterial , tomato, grapevine, tobacco and . Interestingly, of five to six PGIP me mbers, PGIP2 is the most powerful molecule to inhibit polygalacturonase activity secreted by fungal pathogens 39,41. was included as an internal control International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(8), 20-26, August (2013) Int. Res. J. Biological Sci. International Science Congress Association 23 Table-1List of OsKIN13A interacting proteins obtained from yeast two-hybrid screening Category: Transport and binding protein (TB) a Colony number Gene annotation/ putative role 1 Calnexin 1 (CNX1) 11 Dihydrolipoamide S-acetyltransferase (LTA2) 32, 200 Zinc finger (C3HC4-type RING finger) protein b 61 Speckle-type POZ protein 82 Lipid transfer protein-related 85 N-methyl-D-aspartate receptor-associated protein 132 Zinc finger (GATA type) protein 144 FtsJ-like methyltransferase protein 151 Monodehydroascorbate reductase 158 Choline kinase 169 Clathrin adaptor 172 SAR DNA-binding protein-1 181 Selenium-binding protein 218 Glutathione S-conjugate ABC transporter (MRP1) 223 Ubiquitin-conjugating enzyme E2-17 kDa 229 Methyltransferase 230 Glycosyl transferase protein 268 ADP-ribosylation factor 309 Adapter protein SPIKE1 (SPK1) 328 Polyadenylate-binding protein (PABP) 350 Heavy-metal-associated domain-containing protein Category: Metabolic activity (MA) a Colony numberGene annotation/ putative role 5 Endo-1,4-beta-glucanase / cellulase (CEL2) 26 Dienelactone hydrolase family protein 31 Ribulose-bisphosphate carboxylase 43 D-alanine ligase 59 ATP synthase 72 Glyceraldehyde 3-phosphate dehydrogenase 189 Pyruvate decarboxylase 197 Anthocyaninless2 (ANL2) 264 3-hydroxy-3-methylglutaryl-CoA reductase 1 267 Glucan phosphorylase 326 Cinnamyl-alcohol dehydrogenase 329 Glycosyl hydrolase family 17 protein 352 Pyruvate kinase 246 Omega-6 desaturase Category: Cellular processes (CP) a Colony number Gene annotation/ putative role 6 Cyclin-dependent kinase C (CDKC) 22 Pentatricopeptide (PPR) repeat-containing protein 25 Elongation factor 2 (EF-2) 41 Kinesin motor protein 48 Tubulin gamma-1 (TUBG1) 54 Histone H1 70 Hydroxyproline-rich glycoprotein protein 124 Integral membrane protein 127 F-box family protein / SKP1 interacting partner 3-related 136 Beta-expansin 141 Vesicle-associated membrane protein (VAMP) 143 Armadillo/beta-catenin repeat protein 161 Elongation factor 1 (EF-1) 187 Histone H3 194 DEAD box RNA helicase 252 Tubulin alpha-2 269 Actin-depolymerizing factor 280 Disulfide isomerase 343 Histone H4 220 Alpha-tubulin Category: Molecular chaperone (MC) a Colony numberGene annotation/ putative role 35 DnaJ heat shock protein 67, 182 Heat shock cognate 70 kDa protein b 119 FK506-binding protein (FKBP12) / immunophilin 260 Heat shock protein 81-2 (HSP81-2) Category: Signal transduction (ST) a Colony numberGene annotation/ putative role 46 Dehydration-induced protein (ERD15) 73 Calmodulin-7 (CAM7) 78 Serine protease HTRA2 107 SPX domain-containing protein (NUC-2) 110 Wall-associated kinase 4 183 Calcium-dependent protein kinase 191 AP2 domain-containing protein 199 Ethylene-responsive transcriptional coactivator 201, 204 Senescence-associated protein (B12D) b 212 Senescence-associated protein 5 228 Myb family transcription factor 254 3-phosphoinositide-dependent protein kinase 283, 284 Ferredoxin thioredoxin reductase b 310 Transcriptional coactivator p15 (PC4) 313 Floral homeotic protein APETALA1 (AP1) 316 Polygalacturonase inhibiting protein 2 (PGIP2) 341 Nascent polypeptide-associated complex protein Category: Other Colony number Gene annotation/ putative role 126 ARF GTPase-activating domain-containing protein 184 Nodulin 315 Early nodulin-related 321 Endomembrane protein 70 348 Iron-sulfur cluster assembly complex protein Classified according to their function in biological processes.Appeared twice during the Y2H screening. International Research Journal of Biological Sciences ________________________________________________ Vol. 2(8), 20-26, August (2013) International Science Congress Association Structure and phylogenetic A, Molecular structure of OsKIN13A with nineteen exons (gray of motor domain signature (KMD, green color) in OsKIN13A; C, Phylogenetic of OsKIN13A with other kinesins from rice. Classification of OsKIN13A interacting International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association Figure-2 Structure and phylogenetic tree of rice kinesin (Os KIN13A A, Molecular structure of OsKIN13A with nineteen exons (gray - filled boxes) and eighteen introns of motor domain signature (KMD, green color) in OsKIN13A; C, Phylogenetic of OsKIN13A with other kinesins from rice. Figure-3 Classification of OsKIN13A interacting - proteins according to their role in biological processes International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 24 KIN13A ) filled boxes) and eighteen introns (inter-lines); B, Location of motor domain signature (KMD, green color) in OsKIN13A; C, Phylogenetic of OsKIN13A with other kinesins from rice. proteins according to their role in biological processes International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(8), 20-26, August (2013) Int. Res. J. Biological Sci. International Science Congress Association 25 Conclusion This study underlines a critical function of OsKIN13A that involved in wide ranges of biological processes, not only related with cellular mechanism and morphology determination, but also associated with protein folding and plant immunity. 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