Total RNA was isolated using the MasterPure RNA Purification kit (Epicentre Technologies).
rRNA was removed from 100 µg total RNA by annealing rRNA specific primers, extending with MMLV Reverse Transcriptase (Epicentre Technologies) and digesting the RNA-DNA hybrids with RNAse H (Epicentre Technologies).
The 5'-triphosphates of 6 µg total-RNA were converted to monophosphates with 10 units of TAP (Epicentre Technologies) according to manufacturer's specifications.
The ligation and transformation steps were carried out as illustrated in Peterson et al. [16] except that the vector used was HindIII-ready pIndigoBAC5 (Epicentre Technologies).
5' triphosphates were converted to monophosphates by treatment of 15 µg total RNA with 25 units of tobacco acid pyrophosphatase, TAP (Epicentre Technologies).
In EMSA, a core RNAP (50 ng) (Epicentre Technologies, Madison, WI) was incubated on ice for 20 min with 400 ng of purified His6-HrpL protein.
For bacteria and viruses, which were rated for handling in BSL-2 environment or higher rated, inactivated organisms, genomic DNA was extracted in NRL using the MasterPure DNA purification kit (Epicentre Technologies, Madison, WI) according to manufacturer's recommendations.
RNAP (Epicentre Technologies, Madison, WI) and the His6-tagged HrpL (His6-HrpL) were incubated together with digoxigenin (DIG) labeled DNA fragments of hrpA or hrpN promoter regions containing hrp box sequences.
A nylon bristled DNA buccal cell collection brush (MasterAmp™ Buccal Swab Brush, Epicentre Technologies, Madison WI, USA) was then advanced through the catheter until the bristles were just beyond the end of the catheter.
Sequencing reactions were performed with the SequiThermTM Excel Long ReadTM DNA sequencing Kit-LC (EpicenTechnologiesgies) and the DNA sequence of the inserts was determined at the laboratory of Microchemistry, FoRTH, Heraklion (Greece) on a LiCor 4200 DNA sequencer.
The ligation product was directly transformed into competent cell DH10B (Epicentre Technologies, USA) by electroporation.
