Gateway cassette

For LKB1 expression, the complete ORFs of Drosophila Lkb1 (dLkb1) and human LKB1 (hLKB1) were cloned in frame into a P-element based vector that contains the Ubi-p63E promoter and 5’UTR, the GFP coding sequence without stop codon, the Gateway cassette (Invitrogen) placed to allow fusion with the GFP in N-term position and the rosy 3’UTR with a polyadenylation signal [36 (link)]. Injection and transformant selection were performed by Bestgene. At least three independent insertions for all transgenes, excepted dlkb1>GFP-dLkb1 (one insertion), were analysed by western blot and for their rescue ability The results of the most representative are shown. Moreover, for the lines shown in the paper we checked by RT-qPCR on GFP sequence their expression in the testis and they are all in the same range (fold change <2), excepted the one with lkb1 promoter, which was much weaker.
For western blotting, 2-day-old males (n = 15) were dissected in 50μl of lysis buffer, sonicated and boiled for 5 minutes. After centrifugation, 10μl of each supernatant was loaded on precast 4–15% acrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (Biorad) and probed with mouse anti-GFP (dilution) (Ozyme, #JL-8) and anti-tubulin (1/10000) (Sigma #DM1A) antibodies.

Total RNA was isolated from 10-day-old seedlings of T65 using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The SuperScript one-step RT-PCR kit (Invitrogen, Carlsbad, CA, USA) was used for RT-PCR according to the manufacturer’s protocol. The amplification conditions of OsRTFL3 using RT-PCR was one cycle at 95 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min (2720 Thermal Cycler; Applied Biosystems, Foster City, CA, USA). The following pair of primers was used for amplification: OsRTFL3-Fw: 5′-CACCATGGAGGACGAGAGGTGGAAGC-3′ and OsRTFL3-Rev: 5′-CTAGTAGTCTCGCCAGCAGACGAG′. The RT-PCR product was cloned into the pENTRD-TOPO vector (Invitrogen) and then introduced into PH35G, a binary vector containing a Gateway cassette (Invitrogen) in the sense orientation under a CaMV 35S promoter (Narita et al. 2004 (link)).
The construct was introduced into wild-type Arabidopsis using Agrobacterium-mediated transformation with the simplified floral dip method (Clough and Bent 1998 (link)). Transgenic plants were selected on MS medium containing 2 mg mL⁻¹ Gellan Gum (Wako, Osaka, Japan) and 20 μg mL⁻¹ Hygromycin B (Aventis Pharma Ltd., Tokyo, Japan).

Full length cDNAs encoding NOD2 interacting proteins were purchased at the RZPD consortium (Berlin, Germany) or at NITE (Chiba, Japan) and subcloned following PCR in pDONR (Zeo) (Invitrogen) to allow further cloning by recombination in different destination vectors. All cDNAs were fully sequenced. For BRET experiment, cDNAs were cloned into vectors allowing fusion with the donor Renilla luciferase protein or with the EYFP protein (a kind gift of Tarik Issad, France). These vectors were previously modified to facilitate cloning by insertion of a Gateway^™ cassette (Invitrogen Corporation) allowing N-terminus or C-terminus fusion proteins. Similarly the same gateway cassette was introduced in the pEGFP-C1 and pRK5-myc vectors [35 (link)] to express N-terminus EGFP or Myc tagged NOD2, RICK and other proteins.