Ecl direct nucleic acid labeling and detection system

Rice genomic DNA was extracted from 4-week-old rice seedlings using CTAB method as described previously^{64 (link)}. HindIII-digested genomic DNA (30 µg) was separated by electrophoresis on 0.8% (w/v) agarose gel, and blotted onto a nylon membrane (Zeta-probe GT membrane, Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The probe for the hpt gene was isolated from a pIPKB007 vector by PCR amplification. The membranes were pre-hybridized at 65 °C in 7% SDS and 0.25 M Na₂HPO₄ for 2 h and then hybridized overnight at 65 °C in the same solution containing the probe labeled with the enzyme horseradish peroxidase (ECL Direct™ Nucleic Acid Labeling and Detection System, Amersham Biosciences, Piscataway, NJ, USA) for 10–12 h at 42 °C. Membranes were washed twice for 40 min each with 20 mm Na₂HPO₄ and 5% SDS at 65 °C and then washed twice again for 30 min each with 20 mm Na₂HPO₄ and 1% SDS at 65 °C. Finally, the membrane was wrapped in Saran Wrap and exposed to X-ray film (Fuji Film Medical Systems, Stamford, CT) for 1–2 h.

Total genomic DNA was extracted from leaves according to the protocol of Doyle and Doyle [77 ]. Mitochondrial DNA was isolated using the procedure of Horn [78 (link)]. The mitochondrial genes atp6 (subunit 6 of the ATPase gene of sunflower), atp9 (subunit 9 of the ATPase gene of sunflower) and cob (apocytochrome b gene of sunflower) were used as probes after amplification with gene specific primers (Table S3). Primers were designed using web primer (available online: http://www.yeastgenome.org/cgi-bin/web-primer). The probes were labeled, using three different labeling systems: ECL Direct™ Nucleic Acid Labeling and Detection System (Amersham, GE Healthcare, Munich, Germany), ³²P radioactively labeled overgo-primer (Hartmann Analytic GmbH, Braunschweig, Germany) and Prime-It II Random Primer Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the supplier’s instructions.

The M6-lip1^- mutant was screened out of a random transposon library that contains about 10,000 mutants, and was generated in the background of A. citrulli M6 using the Ez-Tn5 kit (Epicentre, Madison, WI, USA), as described [11 (link),16 (link)]. Mutants altered in twitching motility were directly screened on NA/Km selection plates by the naked eye, and verified by colony microscopy observations using an Axio Scope light microscope (Carl Zeiss, Jena, Germany) equipped with a DXM1200F digital camera (Nikon, Tokyo, Japan). The M6-lip1^- mutant was tested by Southern blot hybridization to verify insertion of the EZ-Tn5 cassette, using the ECL Direct Nucleic Acid Labeling and Detection System (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturers’ instructions, and using a region of the EZ-Tn5 cassette as probe [11 (link)]. The insertion site of the EZ-Tn5 cassette was identified following sequencing of the mutant genome by Illumina MiSeq at the Center for Genomic Technologies of the Hebrew University of Jerusalem. Quality trimming and genome assembly were conducted as described [14 (link)].