Rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system

Abstract

Mitogen-activated protein kinase (MAPK) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. Yet, our knowledge of the global physical MAPK interactome in plants remains largely fragmented. Here, we utilized the yeast two-hybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localization, and kinase assay experiments in the model crop rice (Oryza sativa) to systematically map what is to our knowledge the first plant MAPK-interacting proteins. We identified 80 nonredundant interacting protein pairs (74 nonredundant interactors) for rice MAPKs and elucidated the novel proteome-wide network of MAPK interactors. The established interactome contains four membrane-associated proteins, seven MAP2Ks (for MAPK kinase), four MAPKs, and 59 putative substrates, including 18 transcription factors. Several interactors were also validated by experimental approaches (in vivo and in vitro) and literature survey. Our results highlight the importance of OsMPK1, an ortholog of tobacco (Nicotiana benthamiana) salicyclic acid-induced protein kinase and Arabidopsis (Arabidopsis thaliana) AtMPK6, among the rice MAPKs, as it alone interacts with 41 unique proteins (51.2% of the mapped MAPK interaction network). Additionally, Gene Ontology classification of interacting proteins into 34 functional categories suggested MAPK participation in diverse physiological functions. Together, the results obtained essentially enhance our knowledge of the MAPK-interacting protein network and provide a valuable research resource for developing a nearly complete map of the rice MAPK interactome.

Figure 1.

Experimental strategy for establishing a proteome-wide rice MAPK interactome. A, Backbone construction of a MAPK interactome. Rice full-length cDNAs (MAP2K, MAPK, TF, and DRP) in DB and AD vectors were subjected to 2,528 possible combinatorial cross-interactions. Positive interacting protein pairs derived from the first screening gave rise to a MAPK backbone interactome. B, Proteome-wide screening of the rice MAPK interactome. Rice MAPKs and MAP2Ks identified in the backbone interactome were used as baits, and the rice leaf cDNA library was used as prey. Positive interactions from the first screen were confirmed by the retransformation assay in the second screen. Positive interactors from the first and second screens were used to develop a rice MAPK interactome. [See online article for color version of this figure.]

Figure 2.

Genome-wide Y2H screening of the rice MAPK interactors using the rice cDNA library. Results for some of the PPIs are provided as examples. Five tests (lanes 1–5) used to determine the PPIs were the β-galactosidase assay (lane 1) and cell growth on various solid selection media (SC-LT [lane 2], SC-LTH + 25 mM 3-AT [lane 3], SC-LT + 0.2% 5-FOA [lane 4], and SC-LTU [lane 5]). Control tests were performed to assign the qualitative interaction intensity (II) for other interacting protein pairs. Control plasmid pairs are as follows: S (strong), pEXP32/Krev1 + pEXP22/RalGDS-wt; W (weak), pEXP32/Krev1 + pEXP22/RalGDS-m1; A (absent), pEXP32/Krev1 + pEXP22/RalGDS-m2. Krev1 (Rap1A) is a member of the Ras family of GTP-binding proteins. RalGDS is Ral guanine nucleotide dissociation stimulator protein, and its mutants are RalGDS-m1 and RalGDS-m2. Details of all identified MAPK-interacting proteins are provided in Supplemental Table S3. [See online article for color version of this figure.]

Figure 3.

Validation of the Y2H-based identified IPPs by Co-IP and GST pull-down assays. A, Immunoblotting of Co-IP sample using Myc and HA antibodies. In vivo Co-IP was performed using tobacco leaves infiltrated with Agrobacterium C58C1 strain harboring the desired interacting pair (Myc:OsMPK1/HA:OsMEK2). B, Immunoblotting of Co-IP sample using Myc and His antibodies. In vivo Co-IP was performed using tobacco leaves infiltrated with Agrobacterium C58C1 strain harboring the desired interacting pair (His:OsMPK6/Myc:OsMEK2). Symbols represent the presence (+) or absence (–) of the various proteins. C, In vitro GST pull-down assays for 10 interacting pairs. The top panel shows immunoblot analysis of the binding of His-bait fusion proteins (OsMPK1 and OsMPK6) to GST-prey fusion proteins or GST alone (control) using His antibody. Asterisks in the bottom panel indicate the E. coli expression of GST alone and complexes of GST-prey with His-bait fusion proteins purified on columns of Glutathione-Sepharose 4B beads and nickel-nitrilotriacetic acid agarose chelating agarose CL-6B beads, respectively, and subsequently analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Input in A, B, and C was bait fusion protein. IB, Immunoblotting.

Figure 4.

In vivo BiFC analysis of identified IPPs in rice leaf sheaths. Rice leaf sheath was transformed with mixtures of interacting protein pairs using a biolistic method as described in “Materials and Methods.” IPP bait and prey proteins were fused to the N- and C-terminal halves of YFP, respectively. YFP fluorescence and localization were observed by confocal laser microscopy. See text for details. CP, Cytoplasm; NU, nucleus. Bar = 5 µm. [See online article for color version of this figure.]

Figure 5.

Subcellular localization of individual interactors in onion epidermal cells. A, Subcellular localization analysis of OsMPK1 and its eight interacting partner proteins shows the presence of both bait and prey in the cytoplasm and nucleus except OsWRKY80, which was found to be localized only to the nucleus. B to F, Subcellular localization of OsMPK5 (B), OsMPK6 (C), OsWNK1 (D), OsMPK8 (E), and OsbZIP46 (F). G, GFP is shown as a positive control. CP, Cytoplasm; NU, nucleus. Bar = 20 µm. [See online article for color version of this figure.]

Figure 6.

The rice MAPK interactome. The 80 PPIs for MAPK signaling components detected by Y2H screens are shown as an interaction network. Proteins are shown as cubes, hexagons, and circles, and interactions are shown as lines. The interaction pairs for each bait protein are separated by lines of different colors. Kinases are shown as light green and green cubes, hexagons, and circles. TF, regulatory enzyme (RE), metabolic enzyme (ME), and hypothetical protein (HP) are shown in blue, yellow, brown, and purple, respectively. Localization of all of the interacting proteins based on prediction and experimental data are shown. Details of all interaction pairs are given in Supplemental Table S3. [See online article for color version of this figure.]

Figure 7.

OsMPK1- and OsMPK5-mediated responses in organisms. The orthologs of OsMPK1 and OsMPK5 in Arabidopsis, tobacco, human, and yeast are shown, along with their interacting components, leading to different responses. Orthologous proteins are shown in the same colors. Some of the interacting components exhibited strong interactions as per the assay system used. [See online article for color version of this figure.]