The seeds were germinated in pots containing vermiculite and BD nutrient solution  and cultivated at 30°C with a 16 h light period. Bacterial suspensions (108 cfu mL−1) in 10 mM MgSO4 were infiltrated into the abaxial leaf surface of twenty days old V. unguiculata using a syringe without a needle. The plants were kept in a greenhouse at 30°C, illuminated by sunlight and
watered every three days. To CUDC-907 solubility dmso determine the number of endophytic bacteria, ten days after H. rubrisubalbicans infiltration, leaves were superficially disinfected with 70% ethanol for five minutes, washed with sterilized water and homogenized with a sterile pestle and CP-690550 mouse mortar in 1 mL of sterile PBS. Leaf extracts were serially diluted and used to determine the number of bacteria colonizing internal plant tissues by plating on NFbHPN-malate. Oryza sativa L. ssp. japonica seeds (variety BRS Formosa) were surface-sterilized with ethanol 70% for 1 min then shaken in 6% hypochlorite and 0.02% tween 20 for 30 min at 30°C, and washed three times with sterile water. The seeds were germinated in Petri TH-302 dishes containing 1% agar at 25°C for 120 h. Plants were grown in an incubator at 25°C with a 16 h light period and 60% humidity. Thirty seedlings were inoculated five days after germination with 30 mL
of H. rubrisubalbicans strains suspension (108 cfu mL−1) by immersion for 15 minutes. The seedlings were transferred to glass tubes containing 20 mL of Hoagland medium  with 0.2% agar and maintained at 25°C, 16 h light period. The roots were cut 3, 5, 7 and 9 days after inoculation, weighed before surface
sterilization by a 2 minutes wash Selleck Docetaxel with 1% sodium hypochlorite containing 0.01% tween-20, followed by 2 minutes in 70% ethanol, and four washes with sterile distilled water. The samples were then homogenized using a sterile pestle and mortar, and the root extracts diluted in 1 mL of sterile PBS. The number of bacteria colonizing internal plant tissues was determined by plating several dilutions of the extracts on NFbHPN-malate plates. The results reported here represent the average of at least five independent experiments. Recombinant DNA techniques Standard procedures were performed for plasmid DNA extraction, restriction enzyme reactions, cloning and bacterial transformations [60 or according to the manufactures recommendations]. Construction of H. rubrisubalbicans hrpE and hrcN mutant strains The genes hrpE and hrcN of H. rubrisubalbicans in plasmids HR02-MF-00-000-009-C05.km and HR02-MF-00-000-053-F11.km (Monteiro and Petruzziello, unpublished) were disrupted by the transposon EZ:: Tn5TM < TET1 > (Epicentre) that confers resistance to tetracycline. The mutant suicide plasmids were electroporated into the wild type H. rubrisubalbicans strain M1. Recombinant cells were selected for tetracycline resistance and screened for the loss of kanamycin resistance (vector marker).