Supplemental Data; Stanko et al. 2014

List of used primers:

5’-AtMPK10KO: 5'-TCCTGGACCTGGACTTAAACATCCATACT-3'

5'-T-DNA LB-SAL: 5'- TGGTTCACGTAGTGGGCCATCG-3'

3'-AtMPK10.1501: 5'-CAAGTCTATTGCTAAGGGAGGCACGTT-3'

AtMKK2.5': 5’-CCAAATTCCTGACTCAAAGCGG-3'

AtMKK2.3': 5'-GGAAGAGCGGGTGGCGGTTGG-3'

AtACTIN2-5’: 5'-GGAAGGATCTGTACGGTAAC-3'

AtACTIN2-3’: 5’-TGTGAACGATTCCTGGACCT-3’

At3g59790NcoI5': 5'-ACCATGGAGCCAACT AACGATGCTGAGAC-3'

At3g59790NotI3': 5'-CAAAGATATGGGCGGCCGCAATCATTGC-3'

AtMPK10-T218A-1: 5'-CTCCTGAGAGTAATTTAATGGCTGAGTATGTTGTCACAAGATG-3

AtMPK10-T218A-2: 5'-CATCTTGTGACAACATACTCAGCCATTAAATTACTCTCAGGAG-3'

AtMPK10-Y220F-1: 5'-GAGAGTAATTTAATGACTGAGTTTGTTGTCACAAGATGGTACC-3'

AtMPK10-Y220F-2 5'-GGTACCATCTTGTGACAACAAACTCAGTCATTAAATTACTCTC-3'.

AtMPK10promF: 5'-TCTCTTGGGCCCGTTAACATCGGCAAC-3'

AtMPK10promR: 5'-GTCTCAGCATCGTTAGTTGGCTCCATGGT-3'

AtMPK10 in situ F: 5'-TCAATCATTGCTGGTTTCAGGG-3'

AtMPK10 in situ R: 5'-CCAACAGAAGCTCAGGTGCACG-3'

Method: AtMPK10 cloning, agro-infiltration assays, and in situ RNA detection,

An AtMPK10 cDNA was obtained by RT-PCR of RNA isolated from wild-type Col-0 plants. 5'-NcoI and 3'-NotI restriction sides were introduced by PCR by using the primers At3g59790NcoI5' and At3g59790NotI3', respectively. The resulting AtMPK10 cDNA was cloned, sequenced, NcoI/NotI digested, and ligated into a pGREEN binary vector expressing C-terminal HA-tagged proteins regulated by a CaMV 35S promoter (kind gift of A. Schweighofer, University of Vienna). Site-directed mutagenesis (QuickChange, Stratagene) was used to create the two putative AtMPK10 LOF versions. The MAPK activation motif T218EY220 was changed to A218EF220, using the PCR primers AtMPK10-T218A-1 and AtMPK10-T218A-2, AtMPK10-Y220F-1 and AtMPK10-Y220F-2. After sequencing and NcoI/NotI digestion the fragments were ligated into the pGreenII-35S-HA binary vector.

To obtain GFP fusion proteins we transferred NcoI/NotI AtMPK10 cDNA fragments into the Gateway vector pENTR4 (Invitrogen). The resulting pENTR4-AtMPK10 vector was used to transfer the AtMPK10 cDNA into the destination vectors pMDC93 and pBatTL-K -to produce BFP (blue fluorescent protein) and YFP (yellow fluorescent protein) fusion proteins respectively. The localization of the fluorescent proteins was analyzed after agroinfiltration in Nicotiana benthamiana leaves with a Leica-SP1 confocal system as described (Winter et al., 2007b).

The predicted AtMPK10 promoter region of 700 bp upstream of the start codon was amplified by PCR using the primers AtMPK10promF and AtMPK10promR, transferred into a pCR2.1 TA-TOPO vector, sequenced and cloned as a SalI/NcoI fragment into a pENTR4 gateway vector (Invitrogen), and then transfered into the GUS reporter binary gateway vector pKGWS7. All transgenic Arabidopsis mpk10 and Col-0 plants were transformed using the floral dip method as described in Clough and Bent (1998) and selected on MS medium supplemented with Kanamycin or Hygromycin.

In situ RNA detection assays were performed as described (Wahl et al. 2013) by using a synthesized biotinylated sense and antisense RNA fragment synthesized by T7 and SP6 polymerase reactions (Promega) from a pGEM-T vector (Promega) containing a 700bp PCR fragment of the AtMPK10 cDNA (including the start codon; see used oligonucleotides).

References:

Clough, S.J., Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16:735–743.

Wahl, V., Ponnu, J., Schlereth, A., Arrivault, S., Langenecker, T., Franke, A., Feil, R., Lunn, J.E., Stitt, M., Schmid, M. (2013) Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana. Science. 8:339 (6120):704-707.