Glufosinate ammonium

Miscanthus sinensis (identification number: Sin‐13) collected in Honshu, Japan (Clifton‐Brown & Lewandowski, 2002 (link)), were grown from seeds in glasshouse under 8/16‐hr (light/dark) photoperiod at 22–25°C. Arabidopsis thaliana ecotype Columbia‐0 (Col‐0) was grown in soil at 21°C under 8/16‐hr (light/dark) photoperiod until rosette stadium and then transferred to 16/8‐hr photoperiod. Arabidopsis plants were stably transformed by the floral dip method as described (Clough & Bent, 1998 (link)). For selection, plants were grown on half‐strength MS plates supplemented with 25 mg/L hygromycin or 7.5 mg/L glufosinate ammonium (Sigma‐Aldrich); 10‐day‐old transgenic Arabidopsis seedlings of mCherry‐GR‐MsSND1 lines were pretreated with 10 μM cycloheximide (CHX), a protein synthesis inhibitor, for 2 h. To activate mCherry‐GR‐MsSND1, the seedlings were transferred to 10 μM dexamethasone (DEX) and/or 10 μM cycloheximide (CHX) solution for 4 h. Arabidopsis snd1 nst1 double mutant seeds (Mitsuda et al., 2007 (link)) were obtained from the Nottingham Arabidopsis Stock Center (NASC).

The coding regions of GmCPD1, GmCPD2, GmCPD3 and GmCPD4 with additional XbaI and SacI restriction sites were PCR-amplified. The XbaI–SacI flanked GmCPDs fragments were cloned into the XbaI–SacI sites of pTF101.1-GFP vector, replacing GFP and generating pTF101.1-GmCPD1, pTF101.1-GmCPD2, pTF101.1-GmCPD3 and pTF101.1-GmCPD4. These resulting constructs were verified by sequencing and restriction analysis and transformed into Agrobacterium tumefaciens strain GV3101. The Agrobacterium-mediated flower infiltration transformation method [44 (link)] was used to introduce GmCPDs into cpd-91 Arabidopsis mutant plants. T1 generation seeds were harvested and selected on antibiotic-containing MS plates with 10 mg/L glufosinate ammonium (Sigma, St. Louis, MO, USA). Positive plants were confirmed by PCR analysis and propagated to obtain the T3 generation.

The wild type M. oryzae strain Guy11 and its derivative strains were grown on complete medium (CM) plates [20 (link)] at 28°C. Conidia were harvested from 9-day-old cultures grown on CM plates. Genomic DNA and total RNA were extracted from 3-day-old cultures grown in liquid CM. Fungal transformants were screened in CM plates supplemented with 200 μg ml⁻¹ of hygromycin B (Roche Applied Science, Mannheim, Germany) or 250 μg ml⁻¹ of glufosinate ammonium (Sigma-Aldrich, St Louis, MO, USA), depending on the selection marker. Evaluations of conidiation and vegetative growth were performed as previously described [21 (link)]. And growth rate of M. oryzae was determined by measuring the colony diameter of 6-day-old cultures on CM plates supplemented with chemicals.

To overexpress GmCYP82A3, the coding DNA sequence (CDS) of GmCYP82A3 (GenBank: NM_001254043.1) was amplified using the primers CYP82A3-F and CYP82A3-R from Conrad cDNA (S1 Table). The 1584 bp gene fragment was first cloned into the Gateway entry vector pDONR221, then cloned into pEarlyGate202 through an LR recombination reaction between the entry clone and the destination vector (Invitrogen, USA) [41 (link)]. The constructed vector was validated by sequencing, subsequently transformed into Agrobacterium tumefaciens (strain EHA105) by electroporation for further transformation.
The transgenic N. benthamiana plants were generated by A. tumefaciens mediated transformation from leaf discs, as described by Horsch [42 (link)]. The T1 seeds collected from self-pollinated T0 plants were germinated on MS medium with 50 mg/L Glufosinate ammonium (Sigma, USA) to produce T1 transgenic plants. T2 seeds were collected and the plants were cultured for functional characterizations. The transgenic plants were confirmed by PCR screening of both genomic DNA and cDNA using gene specific primers.

MsGSTU8 was ligated with a plant binary expression vector digested by the restriction sites of SacI and PstI to generate the recombinant constructions under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Sham and Aly, 2012 (link)) with bar (phosphinothricin acetyltransferase) as the screening-labeled gene. The recombinant plasmid was transformed into an Agrobacterium tumefaciens strain EHA105 by the freeze-thaw method. The transgenic tobacco lines were obtained with Agrobacterium-mediated leaf disc transformation as described in a previous report (Hannah et al., 2006 (link)). Seeds of the transgenic tobacco lines were sown on MS medium containing glufosinate-ammonium (Sigma-Aldrich) to screen for the positive transgenic lines based on the resistance provided by the phosphinothricin acetyltransferase gene. To further confirm positive transgenic lines, PCR amplification of MsGSTU8 with primer P4 (Table S1) was used, with the extracted genomic DNA as a template. The qPCR was used to investigate the transcript levels of MsGSTU8 of the WT and transgenic tobacco lines, the NtGAPDH gene was used as an endogenous control (Table S1).

The cloned GmBRI1 gene was inserted into pTF101.1-GFP in place of the GFP gene. XbaI and KpnI were chosen as the restriction enzyme cutting sites. The ligated plasmids were transformed into Escherichia coli. The pTF101.1-GmBRI1 plasmids were identified by sequencing and restriction analysis and were transformed into Agrobacterium tumefaciens GV3101. A. tumefaciens containing the pTF101.1-GmBRI1 construct was selected on lysogeny broth (LB) plates containing kanamycin (50 mg/L), spectinomycin (50 mg/L), chloramphenicol (25 mg/L) and rifampicin (20 mg/L). The plasmid was isolated, and the insert was sequenced to confirm the accuracy of the GmBRI1 transgene.
The GmBRI1 transgene was introduced into the bri1-5 mutant Arabidopsis plants by the Agrobacterium-mediated flower infiltration transformation method [43 (link)]. The seeds were harvested from individual plants. Next, the Arabidopsis seeds were sterilized with 50% chlorine bleach solution for 15 min. The seeds were then washed with sterile water 3–5 times and sown on antibiotic-containing MS plates with 250 mg/L carbenicillin, 250 mg/L cefotaxime and 10 mg/L glufosinate ammonium (Sigma, St. Louis, MO, USA). A PCR analysis with primers specific for the CaMV 35S promoter and the GmBRI1 coding sequence was performed to confirm the insertion of the GmBRI1 transgene. T₃ transgenic plants were obtained for investigation.