MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays

Abstract

Signaling through mitogen-activated protein kinases (MPKs) cascades is a complex and fundamental process in eukaryotes, requiring MPK-activating kinases (MKKs) and MKK-activating kinases (MKKKs). However, to date only a limited number of MKK-MPK interactions and MPK phosphorylation substrates have been revealed. We determined which Arabidopsis thaliana MKKs preferentially activate 10 different MPKs in vivo and used the activated MPKs to probe high-density protein microarrays to determine their phosphorylation targets. Our analyses revealed known and novel signaling modules encompassing 570 MPK phosphorylation substrates; these substrates were enriched in transcription factors involved in the regulation of development, defense, and stress responses. Selected MPK substrates were validated by in planta reconstitution experiments. A subset of activated and wild-type MKKs induced cell death, indicating a possible role for these MKKs in the regulation of cell death. Interestingly, MKK7- and MKK9-induced death requires Sgt1, a known regulator of cell death induced during plant innate immunity. Our predicted MKK-MPK phosphorylation network constitutes a valuable resource to understand the function and specificity of MPK signaling systems.

Figure 1.

Comprehensive analysis of in vitro MKK substrate specificity. (A) Combinations of MKK/MPK purified fusion proteins comprising nine wild-type MKK (MKK^WT) and activation loop mutants (MKK^EE) and 10 MPKs, were mixed in 96-well plates with MBP and kinase buffer in the presence of [γ-³²P]-ATP. The proteins were transferred to PVDF membranes and phosphorylated MBP was detected on film. A representative blot is shown. (B) The kinase activity, indicated by phosphorylated MBP, was estimated by measuring the amount of incorporated [γ-³²P]-ATP in all reactions in two or three technical replicates. Spot intensities were obtained from the PhosphorImager exposure. Graphical representation of the likelihood of interaction (−log [P-value]) for the blot in A is shown. The level of significance (0.0143) was represented as the black threshold line at 1.8 U on the Y-axis.

Figure 2.

Generation of Arabidopsis protein microarrays. A representative protein microarray containing 3840 protein preparations printed in duplicates, representing 2158 unique Arabidopsis proteins. The microarray was probed with anti-cMyc monoclonal primary antibody and Cy5-labeled secondary antibody to assess the amount of protein on the slide. For clarity, an enlarged image of one block is shown below the protein microarray.

Figure 3.

Identification of MPK phosphorylation substrates on protein microarrays. (A) Schematic of kinase assays on protein microarrays. MPK-TAP fusion proteins were purified from N. benthamiana overexpressing 20 selected in vitro functional combinations of MKK/MPK. Purified MPK proteins in kinase buffer were overlaid on protein microarrays in the presence of [γ-³³P]-ATP and incubated for 1 h at 30°C. Phosphorylated MPK substrates on the protein microarrays were detected by exposure to Kodak film. (B,C) A representative microarray probed with activated MPK5 (B) and a representative negative control overlaid with kinase buffer and [γ-³³P]-ATP to detect autophosphorylating proteins (C) are shown. Bright dark spots at the corner of each block represents positional controls spotted in duplicate to help with subsequent grid alignment and spot identification. (D,E) Zoomed-in area of block 26 marked as white rectangle from microarrays represented in B and C are shown in D and E, respectively. White rectangle regions in D represent putative MPK5 phosphorylation targets for which signal is present in the MPK5 probed array (D) but absent in the autophosphorylation control array (E).

Figure 4.

Reconstituted weighted MKK/MPK/Substrate phosphorylation networks. (A) The reconstituted network contains nine MKKs, 10 MPKs, and 570 MPK substrates represented as nodes. The size of the nodes is proportional with their degree of connectivity. The network edges indicate identified phosphorylation events. The width of each edge is proportional with the likelihood of the phosphorylation event it represents. The network was generated using Cytoscape version 2.5.1. (B) MKK/MPK/transcription factor hierarchical phosphorylation subnetwork including all predicted WRKY (W) and TGA (T) transcription factors. The nodes represent proteins and the edges represent phosphorylation events. The node size is equal with its degree of connectivity, and edge width is proportional with the likelihood of the phosphorylation event. The network was generated using Cytoscape 2.5.1.

Figure 5.

In planta validation of MPK targets. MKK-HA, MPK-CFP, and WRKY/TGA-TAP proteins were expressed together in N. benthamiana. As negative controls, WRKY/TGA-TAP proteins were expressed alone. Total protein extracts were separated on SDS-PAGE, and blots were probed with anti-cMyc monoclonal antibody. Phosphorylated and nonphosphorylated isoforms of WRKY/TGA factors were detected by mass shifts. Total protein extracts subjected to protein phosphatase 1 or λ phosphatase treatment were run alongside nontreated extracts as controls. Arrows represent phosphorylated protein isoforms.

Figure 6.

Cell death phenotype induced by overexpression of several MKK/MPK modules in N. benthamiana. The diagram on the left represents the position on the leaf and the description of the infiltrated samples. The phenotype of infiltrated leaves shown on the right was monitored for several days post infiltration (dpi). Representative leaf pictures from two consecutive days for each experiment are shown.

Figure 7.

MKK7- and MKK9-induced cell death requires Sgt1. The diagram on the left represents the position on the leaf and the description of the infiltrated samples. MKK7^EE and MKK9^EE were expressed, individually or in combination with cognate MPKs in Sgt1-silenced or VIGS vector-silenced N. benthamiana plants. Representative leaf pictures from two consecutive days for each experiment are shown.