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DOI 10.1002/pmic.201300125

RESEARCH ARTICLE

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system Raksha Singh1∗ , Jae-Eun Lee1∗ , Sarmina Dangol1 , Jihyun Choi1 , Ran Hee Yoo2,3 , Jae Sun Moon2,3 , Jae-Kyung Shim1 , Randeep Rakwal4,5,6 , Ganesh Kumar Agrawal6 and Nam-Soo Jwa1 1

Department of Molecular Biology, College of Life Sciences, Sejong University, Gunja-dong, Gwangjin-gu, Seoul, Republic of Korea 2 Plant Systems Engineering Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 3 Biosystems and Bioengineering Program, University of Science and Technology (UST), Yuseong-gu, Daejeon, Republic of Korea 4 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan 5 Department of Anatomy I, School of Medicine, Showa University, Shinagawa, Tokyo, Japan 6 Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together

Correspondence: Professor Nam-Soo Jwa, Department of Molecular Biology, College of Life Sciences, Sejong University, Gunjadong, Gwangjin-gu, Seoul, Republic of Korea E-mail: [email protected] Fax: +82-2-3408-4336 Abbreviations: 3-AT, 3-amino-1,2,4-triazole; ABA, abscisic acid; AD, activation domain; BiFC, bimolecular fluorescence complementation; BR, brassinosteroid; BRI1, brassinosteriod insensitive 1; BSK1, BR signaling kinase 1; BZR1, brassinazole resistant 1; CTR1, constitutive triple response 1; DMS1, droughthypersensitive mutant 1; EDR1, enhanced disease resistance 1; GFP, green fluorescence protein; GRF10, general regulatory fac-

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: March 27, 2013 Revised: October 27, 2013 Accepted: November 6, 2013

tor 10; ILA1, increased leaf angle 1; IPP, interacting protein pair; KEG, keep on going; KOME, Knowledge-based Oryza Molecular Biological Encyclopedia; MAPK, mitogen-activated protein kinase; MAP2K, mitogen-activated protein kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogenactivated protein kinase kinase kinase; NR, nonredundant; NPK1, Nicotania tabacum protein kinase 1; OsEDR1, Oryza sativa enhanced disease resistance 1; PPI, protein–protein interaction; SCLTH, SC lacking leucine–tryptophan–histidine; YPD, yeast extract peptone dextrose; Y2H, yeast two hybrid ∗ These

authors contributed equally to this work. Colour Online: See the article online to view Figs. 1–5 in colour.

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involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. Keywords: Interactome / MAP3Ks / Plant proteomics / Protein–protein interactions / Rice / Translational proteomics / Yeast two hybrid

 1

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

The mitogen-activated protein kinase (MAPK) signaling pathway is highly conserved in eukaryotes from yeast to mammals as well as in plants [1–3]. A typical pathway is organized into a classic unit of a hierarchical MAP3K (MAPK kinase kinase)/MAP2K/MAPK module (where MAP2K is mitogen-activated protein kinase kinase) [1]. In mammals, the importance of the MAPK signaling pathway became apparent through their involvement in dynamic cellular processes, such as gene expression, cell proliferation, cell survival and death, and cell motility [4]. In plants, MAPK cascades were shown to regulate a diverse array of essential signaling pathways, including responses to various abiotic and biotic stresses [2, 5–8]. However, the complete MAPK cascades studied in plants are far fewer than those identified in yeast and mammals [9–12]. Two examples of the identified complete MAPK cascade in Arabidopsis are MEKK1–MKK4/MKK5–MPK3/MPK6–WRKY22/WRKY29 u pon flagellin perception by flagellin receptor FLS2 and YDA-MKK4/MKK5-MPK3/MPK6, which regulates stomatal development [13, 14]. However, genetic studies by Ichimura et al. and Suarez-Rodriguez et al. showed that AtMEKK1 is dispensable for flg22-triggered activation of AtMPK3 and AtMPK6, indicating that functionally redundant MAPKKKs to MEKK1 are involved in activation of AtMPK3 and AtMPK6 [15, 16]. In plants, more than 60 predicted MAP3Ks, ten MAP2Ks, and 20 MAPKs were identified in Arabidopsis, along with 75 MAP3Ks, eight MAP2Ks, and 17 MAPKs in rice [3,17–20]. In Arabidopsis, MAP3K was reported as a huge family with many members [17]. The 75 rice MAP3Ks were predicted based on the phylogenetic relation to Arabidopsis MAP3Ks [19]. Both rice and Arabidopsis MAP3Ks are classified into three distinct groups (A, B, and C) with two distinct subfamilies based on the kinase catalytic domain [17, 19]. Group A contains the MEKK family, whereas groups B and C contain the Raf-like family [17, 19]. However, to date, the exact number of functional MAP3Ks is not known in all plant species.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In recent years, some of the individual MAP3Ks have been studied in different plant species. The first identified plant MEKK was NPK1 (Nicotiana tabacum protein kinase 1), which is known to regulate cell division [21–23]. Two Raf-like MAP3Ks, CTR1 (constitutive triple response 1) and EDR1 (enhanced disease resistance 1), were reported to be involved in negative regulation of ethylene response and salicylic acid inducible defense response, respectively, in Arabidopsis [24, 25]. In rice, Oryza sativa enhanced disease resistance 1 (OsEDR1) was the first reported MAP3K in the regulation of defense responses [26, 27]. Two more Raf-like MAP3Ks, DMS1 (drought-hypersensitive mutant 1) and ILA1 (increased leaf angle 1), were reported in rice in the regulation of drought stress and mechanical tissue formation, respectively [28, 29]. Despite their large numbers, very few MAP3Ks have been characterized, which raises questions as to whether some of them are pseudogenes or if they are expressed as functional genes. Recently, Ning et al. reported the yeast two hybrid (Y2H) screening of six ILA1 non-MAPK interactors, which regulate mechanical tissue formation [29]. Apart from ILA1, no further information is available, including the upstream and downstream interactors of rice MAP3Ks. To uncover the complexity of the MAPK signaling pathway, it is crucial to outline the regulatory network of upstream and downstream interacting protein pairs (IPPs) involved in corresponding cellular functions. The IPPs function as regulatory molecules in signal transduction pathways relevant to a variety of abiotic and biotic stress responses, hormonal regulation, and defense responses [30]. Therefore, the main goal of our study was to construct a primary rice MAP3K network using expressed forms of 12 full-length MAP3K cDNA clones from the Knowledge-based Oryza Molecular Biological Encyclopedia (KOME) cDNA library of 75 predicted putative sequences. Most of the annotated forms of rice MAP3K candidates were selected from the rice genome sequence database using Arabidopsis MAP3Ks. Twelve full cDNA rice MAP3K clones were identified and distributed from the KOME database. Extensive Y2H screening of the www.proteomics-journal.com

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rice cDNA library was first performed to identify potential MAP3K interactors; this was followed by in vivo bimolecular fluorescence complementation (BiFC) confirmation, subcellular localization analysis, and a literature survey. The findings of this study provide new insights into the members of MAP3Ks interactors, which allowed us to revisit the MAPK cascade and its model. The developed rice MAP3Ks interactome proved a useful resource, as exemplified by comparing it with the MAP3Ks interactomes of Arabidopsis and humans. As MAPK cascades have broad responses to biotic and abiotic stresses, the importance of this interactome study is discussed as translational research relevant to crop improvement. This is the first report of the rice MAP3K interaction network by Y2H screening, and this primary rice MAP3K network will help to predict the broader network and functional roles of these signaling cascades at the MAP3K level.

2

Materials and methods

2.1 DNA constructs The rice cDNA clones were obtained from the Rice Genome Resource Center (Tsukuba, Japan). The full-length ORFs of MAP3Ks were amplified using the primers AttB1 (5 ACAAGTTTGTACAAAAAAGCAGGCT-3 ) and AttB2 (5 ACCACTTTGTACAAGAAAGCTGGGT-3 ). Amplified PCR products were used as substrates to amplify 12 MAP3Ks using their gene specific primers (Supporting Information Table 1). Amplified MAP3Ks were then cloned into the pDONR vector using the Gateway BP clonase recombination TM system (ProQuest Two-Hybrid System, Invitrogen; catalog number 10835), followed by confirmation of their nucleotide sequence. The ORF for each resulting “entry” clone was then integrated into both the pDEST32 and pDEST22 TM Gateway-compatible “destination” vectors (ProQuest TwoHybrid System) to generate the (MAP3Ks:pDEST32; bait) and (MAP3Ks:pDEST22; prey) fusion constructs, respectively, as described previously [31]. The MAP3Ks in the pDONR vectors were subcloned into the BiFC vector containing the Cterminal (pDEST-VYCE(R)GW ; bait) and N-terminal (pDESTVYNE(R)GW ; prey) Venus fusion using the Gateway system, as described previously [32]. For subcellular localization analysis, the MAP3Ks in the pDONR vectors were subcloned into pGWB552 vector containing the N-terminal G3-GFP (where GFP is green fluorescence protein) using the Gateway system as described previously [33].

2.2 Rice cDNA library construction A rice (Oryza sativa japonica cultivar, Dongjin) cDNA library was constructed from 3-week-old seedlings as described previously [31].  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.3 Y2H screen The Y2H screen was performed as described previously [31]. Briefly, autoactivation of all bait was checked. For this, (MAP3Ks:pDESTTM 32) bait was co-transformed with empty vector (pDEST22) in the yeast strain MaV203 (Invitrogen), followed by selection of yeast cells on SC-LT plates (synthetic complete lacking leucine-tryptophan; 6.7 g/L yeast nitrogen base without amino acids, 0.64 g/L SC-LT, 20 g/L agar, 20 g/L glucose) at 30⬚C for 4 days. Individual colonies were picked from the plates and cultured in 1 mL of SC-LT broth at 30⬚C overnight. The cultured cells (OD at 600 nm = 0.2) were spotted on selection plates (SC-LTH (SC lacking leucine–tryptophan–histidine) with 0, 10, 25, 50, 75, or 100 mM 3-AT (3-amino-1,2,4-triazole), and yeast extract peptone dextrose (YPD)) at 30⬚C for 4–5 days to monitor the activation of two reporter genes, HIS3 and LacZ. The concentration of 3-AT on which bait did not exhibit growth was used for library screening. The cultured cells were also spread on SC-LTH plates containing 55 mM 3-AT and grown at 30⬚C for 7 days. The average transformation efficiency was 2.9 × 106 CFU/␮g. Individual colonies that appeared until 7 days were further streaked on SC-LT plates and grown at 30⬚C for 3 days. Each colony was then cultured in 1 mL of SC-LT broth at 30⬚C overnight. The cultured cells (OD600 = 0.2) were spotted on selection plates containing SC-LTH with 0, 10, 25, 50, 75, or 100 mM 3-AT; SC-LT; SC-LTU (U, uracil); SC-LT with 0.2% v/v 5-FOA (5-fluoroorotic acid); and YPD at 30⬚C for 5 days with the exception of the YPD plates (1 day). A ␤-galactosidase assay was performed according to the supplier’s protocol (Invitrogen) and as described previously [31]. A retransformation assay was also performed according to the supplier’s protocol to eliminate false-positive IPPs (Invitrogen). Isolated positive plasmids were subjected to nucleotide sequencing analysis to confirm gene identities (Macrogen).

2.4 Bimolecular fluorescence complementation Onion tissue preparation, DNA preparation, cotransformation, and biolistic bombardment were carried out as previously described [34]. For co-transformation, 8 ␮g each of bait protein in pDEST-VYCE(R)GW and prey protein in pDEST-VYNE(R)GW BiFC vectors were bombarded in R onion epidermal cells by biolistic bombardment (Biolistic PDS-1000/He Particle Delivery System; Bio-Rad) [31]. For co-transformation of OsMEKK11 + OsGRF, OsMEKK11 + OsEP2, OsMEKK24 + OsFHA1, and OsMEKK24 + OsFHA2, 12 ␮g each of bait and prey protein were bombarded in onion epidermal cells. Co-transformed onion cells were incubated at 25⬚C for 12–24 h on a 1/2 MS plate, followed by microscopic observation by a 20× objective lens using an excitation wavelength of 488 nm (argon laser) and an emission wavelength of 505–550 nm on a confocal laser microscope (TCS SP5; Leica). www.proteomics-journal.com

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Table 1. Summary of rice MAP3K IPPs identified by Y2H screening and BiFC assay

Baits (DB domain)

Preys (AD domain)

Y2H interaction

BiFC

Total interactors

Total interactome

OsMEKK8 OsMEKK10 OsMEKK11

OsAPK1 N/D OsEP2 OsPep OsGRF OsDegp6 N/D N/D OsMEKK24 OsFHA1 OsFHA2 OsTY3 OsEP1 OsRingE3 N/D OsDegp10 N/D N/D

++

N/C

1

2

++++ ++++ +++++ +++++

+++ ++++ +++ N/C

3

4

1

2

+++++ ++++ ++++ ++++ ++++ ++++

N/C +++ +++ N/C ++++ N/C

4

5

2

3

+++++

+++++

1

2

OsMEKK16 OsMEKK19 OsMEKK22 OsMEKK24

OsMEKK25 OsMEKK28 OsMEKK55 OsMEKK62 OsMEKK65

MAP3Ks, mitogen-activated protein kinase kinase kinase; MAPKK, mitogen-activated protein kinase kinase; N/D, not detected; and N/C, not checked.

2.4 Subcellular localization Onion epidermal cells were used for subcellular localization analysis of the identified interactors as described previously [34]. All images were observed by a 20× objective lens under GFP filter using confocal laser microscopy (TCS SP5; Leica).

2.5 Functional annotations Functional annotations were done on the basis of GO terms from the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/).

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Results and discussion

3.1 Identification of novel IPPs of 12 rice MAPKKKs To draw the basic networking frame of the MAP3K interactome, we carried out Y2H screening using 12 rice fulllength MAP3Ks, which were available as expressed fulllength cDNA clones at the time of experimental design and execution from KOME (http://cdna01.dna.affrc.go.jp/ cDNA/CDNA_main_front.html). Ten MAP3Ks from group A (MEKK) and two MAP3Ks from group B (Raf-like) were used as bait (Supporting Information Fig. 1 and Supporting Information Table 2). Their division into groups A (MEKK) and B (Raf-like) was based on the sequence alignment of the 12 rice MAP3Ks with those proteins of other plants (Arabidopsis thaliana, Zea mays, Hordeum vulgare, Nicotiana benthama C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

nia, Lycopersicon esculentum, and Brassica napus; Supporting Information Fig. 1). Twelve MAP3Ks were cloned into Y2H destination vectors to generate GAL4 DNA-binding domain or DNA-activation domain (AD) fusion proteins, referred to as DB bait or AD prey, respectively (Supporting Information Table 2). Autoactivation test of 12 baits did not activate the reporter genes LacZ and HIS3, showing that MAP3K baits do not activate the empty vectors on their own (data not shown). Screening of 12 baits gave rise to 12 IPPs, representing 12 nonredundant (NR) interactions (Table 1 and Supporting Information Table 3). The IPPs were further divided into strong and weak interactions based on their interaction intensity. Strong IPPs showed intense blue color on the ␤-galactosidase assay (LacZ) and growth on SC-LTH (HIS3) and SC-LTU (URA) within 2– 4 days, whereas weak IPPs had a longer incubation time. Of 12 IPPs, only one weak protein–protein interaction (PPI) pair (OsMEKK8 + OsAPK1) was identified for OsMEKK8. In addition, strong IPPs were identified for OsMEKK11 (three IPPs), OsMEKK16 (one IPPs), OsMEKK24 (four IPPs), OsMEKK25 (two IPPs), and OsMEKK55 (one IPP; Fig. 1A and Table 1). We found no interacting partner using OsMEKK10, OsMEKK19, OsMEKK22, OsMEKK28, OsMEKK62, and OsMEKK65. All of the positive interacting pairs were further confirmed by a retransformation assay to remove possible false positives in a second round of screening according to the methods described in Section 2 (data not shown). Identified interacting clones were checked for in-frame Gal4 AD fusion by DNA sequencing (Macrogen). The size of the positive clones varied from 500 to 1200 bp. In total, the cDNA library screening of 12 MAP3K baits revealed 12 PPI pairs (Supporting Information Table 3). All of the identified Y2H interactors (100%) are novel with no previous reports. www.proteomics-journal.com

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Figure 1. Identification and validation of MAP3Ks IPPs. (A) Y2H screening of the rice MAP3K baits using a rice cDNA library as prey. The interactions were verified by five tests {␤-galactosidase assay and cell growth on various solid selection medias like [SC-LT (+5-FOA, 0.2%), SC-LTU (Uracil), SC-LTH (+50 mM 3-AT), and SC-LT]}. Interaction intensity (II) of the IPP, which was determined by the color intensity and growth pattern in ␤-galactosidase assay and various solid selection media as in Section 2, are shown by a (+) sign. Strong interactions are shown by (++++) and (+++++), whereas weak interactions are indicated by (++). (B). In vivo BiFC analysis of Y2H-detected IPPs in onion epidermal cells. Onion epidermal cells were transformed with mixtures of interacting protein pairs using a biolistic method as described in Section 2. Interacting bait and prey proteins were fused to the C- and N-terminal halves of Venus, respectively. Venus fluorescence and localization were observed by confocal laser microscopy. [pEXP-VYCE(R)-CnX7 + pEXP-VYNE(R)-CnX6] pair and empty vector pair (pDest-VYNE(R)GW + pDest-VYCE(R)GW ) were used as positive and negative controls, respectively. See text for details.

3.2 Validation of the Y2H-based MAP3Ks interactome by in vivo BiFC and subcellular localization analysis The BiFC approach was applied to subsets of identified IPPs to assess whether the observed Y2H PPIs occur in living plant tissues/cells. This approach has been used successfully to strengthen the Y2H-based rice MAPK interactome data [31, 32]. Of 12 PPI pairs, we could express seven representative pairs (58.3% of total) by BiFC assay under our lab conditions. Arabidopsis CnX6 and CnX7 reportedly form complex heterodimers and are suitable for positive control in a BiFC assay [32]. Therefore, onion cells co-expressing pEXP-VYNE(R)-CnX6 and pEXP-VYCE(R)-CnX7 were used  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

as positive controls and cells co-expressing bait and prey protein with empty pDEST-VYCE(R)GW and pDEST-VYNE (R)GW were used as negative controls (Fig. 1B, Supporting Information Fig. 4A–C). As expected, the CnX6/CnX7 manifested a fluorescence signal of the Venus fluorescence protein and was localized throughout the cytoplasm and nucleus (Fig. 1B). Co-expression of (OsMEKK25 + OsEP1) resulted in a strong Venus fluorescent protein signal only in the nuclear region (Fig. 1B). By contrast, co-expression of (OsMEKK55 + OsDegp10) and (OsMEKK11 + OsPep) was detected both in the cytoplasm and the nucleus (Fig. 1B). Additionally, co-expression of (OsMEKK11 + OsGRF) and (OsMEKK11 + OsEP2) resulted in Venus signal only in the plasma membrane (Supporting Information Fig. 3).

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Figure 2. The primary frame for the rice MAP3K interactome. Twelve IPPs for MAP3K baits detected by Y2H screens are shown in an interaction map. The interacting proteins for each bait protein are separated by lines of different colors. MAP3K baits are shown with a light green color. All of the interacting proteins were localized to the respective compartment based on the information given by the KOME database (http://cdna01. dna.affrc.go.jp/cDNA/CDNA_main_ front.html). Detailed information for the 12 IPPs is given in Supporting Information Table 3.

However, co-expression of (OsMEKK24 + OsFHA1) resulted in Venus signal both in plasma membrane and nucleus whereas co-expression of (OsMEKK24 + OsFHA2) gave rise to Venus signal throughout the cytoplasm (Supporting Information Fig. 3). To address false-positive interactions, each interacting protein of these seven IPPs were tested for its interaction with negative control BiFC vector carrying half of the Venus (Supporting Information Fig. 4A–C). None of them showed fluorescence signal. Taken together, these BiFC data indicate that PPIs detected by the Y2H screen also interact in vivo, which further supports that the remaining PPIs could also interact in vivo. For biologically relevant interactions, both of the interacting proteins usually localize to the same cellular compartment or an interactable compartment [35]. Therefore, we examined the subcellular localization of 14 interactors (nine IPPs) of 18 interactors (12 IPPs) including empty vector (pGWB552) as a positive control by transiently expressing them as Nterminal G3-GFP fusion proteins in onion epidermal cells (Supporting Information Fig. 5A–F) [33]. OsMEKK8, OsMEKK24, OsMEKK25, and OsMEKK55 were localized to the plasma membrane and nucleus and their interacting proteins also localized to the same compartment (Supporting Information Fig. 5B and D–F). In some case, both of the interacting proteins localized to different cellular organelles and under certain physiological conditions one of the interacting protein translocate to the same cellular organelle occupied by its interacting protein [36]. In correlation with this statement, OsMEKK11 interactors localized to the different cellular organelle than that of their baits. OsMEKK11 localized to the plasma membrane only but its interactors OsEP2 localized to the plasma membrane and nucleus; OsGRF localized to the plasma membrane, cytoplasm, and nucleus; and OsPep localized to the nucleus only (Supporting Information Fig. 5C). These results further suggest the possibility of biological relevance of the identified IPPs.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.3 Functional annotations of rice MAP3K-interacting proteins We constructed a rice MAP3K interaction map consisting of 12 interactors by Y2H screening with in vivo validation of seven IPPs by BiFC (58.3%; 7 of 12 IPPs; Fig. 2) and nine IPPs by localization analysis (75%; 9 of 12 IPPs; Fig. 2). Among 12 interactors, two were kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein (Supporting Information Table 3). These 12 interacting proteins were further characterized by analyzing their respective functional annotations using the GO annotation of the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/) into 20 different functional categories with significant enrichment of proteins involved in a number of processes including kinase, metabolic, protein modification, catalytic activity, cellular and protein metabolic process (Supporting Information Fig. 2). GO annotation was not available for two interacting proteins, OsTy3 and OsEP2, during the preparation of this manuscript. Besides these, most of the proteins have several functions, and they were classified into more than one category. For example, OsAPK1 (OsMEKK8-interacting protein) was placed into ten functional categories, whereas OsRingE3 (OsMEKK25-interacting protein) and OsMEKK24 (OsMEKK24-interacting protein) were placed in eight functional categories (data not shown). The highest numbers of proteins were counted for kinase activity and metabolic process (9.30%), protein modification process, catalytic activity, cellular process, protein metabolic process, biological process, and protein binding (6.97%; Supporting Information Fig. 2). Few proteins were involved in nucleotide binding, response to endogenous stimulus, signal transduction, binding and hydrolase activity (4.65%), and various other functional categories as shown in Supporting Information Fig. 2. These www.proteomics-journal.com

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Figure 3. Putative functional predictions of MAP3K-interacting proteins identified in this study compared with studies in Arabidopsis. Orthologous proteins are shown in the same colors. Interaction between two proteins is indicated by different color lines based on the type of approach that was used to identify the interaction.

data would contribute to the dissection of putative roles of the MAP3K IPPs.

3.4 Discussion Construction of a PPI map is the first step toward the system biology of a given organism [37]. The PPI network not only provides clues for the signaling pathway but also helps to consider protein functions. The established interactome consists of 12 NR IPPs and 16 NR protein interactors. Our study identified novel potential interactors (100%) previously not reported in rice. Seven (58.3%) and nine (75%) IPPs of the interactome were validated in vivo by BiFC and localization analysis, respectively, which further support that our remaining PPIs could also interact in vivo, but unfortunately could not be expressed under our lab conditions. The classic MAPK cascades are composed of MAPKs/MAP2Ks/MAP3Ks, and a certain MAP2K is supposed to interact with upstream MAP3Ks. However, our results suggested that all the MAP3Ks may not follow the classic MAPK cascades. We could find no interactions by a pairwise interaction test with all rice MAP2Ks and MAPKs (data not shown) and using a rice cDNA library as prey [31]. Possible explanations exist for this disagreement. First, most of the MAP3Ks might facilitate indirect interactions with MAP2Ks or MAPK with the help of adapter proteins. A second possibility is that they might have direct regulatory functions with currently identified interacting proteins such as OsAPK1 (OsMEKK8 + OsAPK1), OsGRF (OsMEKK11 + OsGRF), OsDegp6 (OsMEKK16 + OsDegp6), and OsRingE3 (OsMEKK25 + OsRingE3), whose orthologs are well studied in Arabidopsis [38–44]. Our hypothesis may be supported by other studies on rice MAP3Ks in which MAP3K-interacting partners other than MAPKKs were found [29]. Several possible canonical MAP3K/MAP2K/MAPK signaling pathways have been proposed. Unfortunately, however, during the design of the experiment, most of the MAP3Ks were not available as expressed full-cDNA clones as predicted (http://  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

cdna01.dna.affrc.go.jp/cDNA/CDNA_main_front.html), although 75 rice MAP3Ks were predicted and reported by annotation [19]. Thus, further characterization of expressed forms of additional rice MAP3K cDNAs should be undertaken to achieve an appreciable size of rice MAP3K PPI screenings. However, the possibility exists of a less complex MAP3K interaction network, and additional study is needed for reconfiguration of the whole rice MAP3K interactome. As mentioned before, little or no information is available on the functional characterization of the identified 12 novel interactors in rice. To determine whether orthologs of these interactors are functionally characterized, we identified four Arabidopsis proteins, BR signaling kinase 1 (BSK1), general regulatory factor 10 (GRF10), KEG (keep on going), and DegP2, for the rice interactors OsAPK1, OsGRF, OsRingE3, and OsDegP6, respectively (Fig. 3). These Arabidopsis proteins were shown to control the responses of brassinosteroid (BR) signaling, abscisic acid (ABA) signaling, and photosystem II. The Arabidopsis BSK1 was previously shown to activate the BR signaling downstream of the receptor BRI1 (where BRI1 is brassinosteriod insensitive 1) [45,46]. Another protein GRF10 was also involved in BR signaling [47]. The GRF10 encodes a 14-3–3 protein and interacts with the brassinazole resistant 1 (BZR1) TF. Their interaction may affect BZR1 shuttling between the nucleus and cytoplasm [47]. Furthermore, GRF10 was proposed to function as a regulatory protein (perhaps scaffold) as it was found to be active in various signaling complexes [48]. The Arabidopsis KEG was reported to interact with ABI5 (ABA insensitive) TF in vitro and is required for ABI5 degradation in ABA signaling [49,50]. DegP2 was found to be involved in stress-related degradation of photosystem II light-harvesting protein Lhcb6 [51]. In light of these reports, the identified rice MAP3K interactors are likely involved in multiple and essential signaling pathways. Recently, a rice E3 ligase was shown to be involved in PAMP-triggered immunity through interaction with fungal effector protein AvrPiz-t [52]. OsMEKK25’s interaction with OsRingE3 connotes its direct involvement in plant defense and raises the possibility of a trimer complex formation with www.proteomics-journal.com

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Figure 4. Comparisons of MAP3K interactome patterns based on physical interaction among various organisms. Identified MAP3K interactome is compared with the reported MAPK interactome of Arabidopsis and humans [9, 10]. Orthologous proteins are shown in the purple color dashed circle.

AvrPiz-t. This means that OsMEKK25 may play a crucial role in PAMP-triggered immunity through interaction with OsRingE3, and that this signaling pathway can be interrupted by fungal effector protein AvrPiz-t as previously hypothesized [53]. OsMEKK25-interacting protein OsRingE3, an ortholog of Arabidopsis KEG, has been studied by several research groups in ABA and defense signaling [41–44]. From this, we can hypothesize that (OsMEKK25 + OsRingE3) may be involved in both PAMP-triggered immunity and ABA responses in rice as well. In addition to the above-mentioned interactions, we could not ignore the possibility of adding unknown signaling pathways as indicated by the rest of the IPPs whose orthologs are not well studied. Apart from the possibility driven by ortholog studies, GO classification of the interacting proteins into 20 functional categories provides strong evidence for the potential involvement of rice MAP3Ks in diverse physiological responses as expected from the currently reported rice MAPK interactome (Fig. 2) [31]. The biological significance of identified interactions ultimately depends on addi-

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tional experimental setups, such as a phosphorylation assay and phenotypic study of gain-of-function and loss-of-function mutants. Therefore, follow-up experiments are necessary to confirm the detected interactions and to evaluate their physiological relevance. These initial results provide us with a valuable rice reference MAP3K interaction network, which we can extend further down to MAP2Ks and MAPKs through individual characterization of currently available interactome components by diverse approaches. The identified MAP3K interactome can be compared with reported MAPK interactomes from Arabidopsis and humans [9, 10]. Rice, Arabidopsis, and humans share similar MAP3K interaction patterns. Only one MAPK cascade is identified in Arabidopsis by physical interaction, whereas in rice and humans, no MAPK cascade is identified, as marked in the figure (Fig. 5). We screened rice orthologous proteins from the Arabidopsis MAPK cascade. In contrast to Arabidopsis, we could find no interacting protein for OsMEKK28 (ortholog of Arabidopsis AtMEKK1) (data not shown). However, many PPIs were observed between rice MAP2K and MAPK orthologs.

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Figure 5. Possible MAPK cascade model based on different approaches in rice. (A) Classical MAPK cascade module. (B) Possible MAPK cascade based on physical interaction. Interaction between several MAP2Ks and MAPKs is identified on the basis of physical interaction [31]. No interaction is identified between MAP3Ks and MAP2Ks or MAPKs on the basis of physical interaction. (C) Activity-based MAPK cascade. In Arabidopsis, the full MAPK cascade is identified on the basis of physical interaction and activity assays [15, 16, 55– 57]. (D) Proposed MAPK cascade based on scaffold binding. The broken arrow represents an unknown possible interaction.

These results indicate that the orthologous proteins from any two organisms do not necessarily interact. Most of the MAP3K-interacting proteins identified in all of these organisms (rice, Arabidopsis, and humans) are regulatory proteins and transcription factors. If we look at the previously reported Arabidopsis and human MAP3K interaction patterns, they are also found to interact mostly with transcription factors (35.9% in Arabidopsis and 12.8% in humans), regulatory proteins (7% in Arabidopsis and 38.5% in humans), and various other proteins as shown in Supplementary Information Table S4. These data show that the MAP3K interaction patterns in rice, Arabidopsis, and humans are somewhat similar (Fig. 4). We know that dimer formation is essential for MAPKKK phosphorylation and activation [54]. Dimerization between MAP3K is shown by some MAP3Ks identified in the rice MAP3K interactome and also by previously reported Arabidopsis and human MAP3K interaction patterns (Fig. 4). In rice, OsMEKK24 forms dimers, similar to Arabidopsis RAF17 and human MAP3K10. Further analysis of the physical interaction of all the identified MAP3Ks will help clarify the MAPK cascade, especially MAP3K-interacting proteins. In Arabidopsis, the full MAPK cascade is reported on the basis of physical interaction and activity assays (Fig. 5), but until now, no MAPK cascade had been reported in rice on the basis of physical interaction or by activity assay [15, 16, 55–57]. To the best of our knowledge, no previous report has described MAPK or MAP2K interacting with MAP3Ks (Fig. 5). Absence of interacting MAPK or MAP2K creates the possibility of several other unknown factors, such as binding specificity, scaffold protein association, and protein conformation and protein stability. As we identified MAP3K-interacting proteins other than MAPKs and MAP2Ks, a possibility exists  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

that these proteins can act as scaffold proteins that tether MAPK components in one complex (Fig. 5). Hence, it will be interesting to further clarify the exact interacting mode or mechanisms of these MAPKs, MAP2Ks, and MAP3Ks by physical interaction.

4

Concluding remarks

This study presents a Y2H screening of 12 novel rice MAPKKKs (bait) with a rice cDNA library as prey using transcriptionally expressed full cDNA clones from a rice cDNA bank. Using this system, we found 12 novel PPI pairs and established the first frame for the rice MAPKKK interactome (Fig. 2). Among 12 PPI pairs, only one pair (OsMEKK8 + OsAPK1) was identified as a weak IPP, and the others were identified as strong IPPs. The Y2H interactions of seven (58.3%) and nine (75%) IPPs were further confirmed in vivo by BiFC and subcellular localization analysis, respectively. These BiFC and subcellular localization data further support that Y2H IPPs also interact in vivo. To examine the functional significance of the above findings, we classified the 12 IPPs into 20 functional groups based on GO classification. The GO classification indicates the involvement of MAPKKKs in diverse physiological responses. Our Y2H-screened IPPs are small in size because of the limited number of available expressed MAP3K cDNA clones, but they are novel and not well characterized in rice. However, the orthologs of four MAPKKK-interacting proteins (OsAPK1, OsGRF, OsDegp6, and OsRingE3) are well characterized in Arabidopsis, which will help unveil the as yet unknown biological roles of MAPKKK signaling in rice. www.proteomics-journal.com

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This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center; No. PJ008061), Rural Development Administration, Republic of Korea, and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant number: 2013R1A1A2009269). The authors have declared no conflict of interest.

5

References

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