Samples were treated with DNase I (Invitrogen) according to the manufacturer’s instructions, and then stored at -80°C until use. To obtain RNA from cells growing in the host, at least 20 citrus leaves were infiltrated with a suspension of Xcc 306 cells (OD 0.3, 600 nm). At 3 days after inoculation, leaves were collected and minced in cold distilled water, in order to facilitate the exudation of bacterial cells to the liquid medium. After 10 min of agitation in an ice bath, the cut leaves were removed and bacterial cells were collected in a Corex tube by centrifuging at 5,000 × g for 10 min. Total RNA extraction and

DNase I treatment were perfomed as described above. Eleven primer pairs (Table 1) were designed for the amplification of the 11 Xcc ORFs for which some sort of virulence deficiency was detected after mutation. The amplification products were used in a nucleic acid selleck chemical hybridization using labeled cDNA probe technique as described below in order to assess possible differential

gene expression in these mutants. NVP-BGJ398 concentration Table 1 Primers used in nucleic acid hybridization. Primers and respective Xanthomonas citri subsp. citri ORFs employed in the amplification of ORFs used in nucleic acid hybridization using labeled cDNA probes. ID ORF Size (bp) Forward Primer Reverse Primer 1 XAC0340 432 gATACCCCATATgAATgCgAT CAgCgCCAAgCTTATgCCATg 2 XAC0095 222 AggAgAgCCATATgCACgACg TTgCATCgAATTCAgTgCgTT 3 Water       4 XAC1927 1.179 ggAgTCTCATATgCTgACgCg CCggTACCTCgAgTgTCATg 5 XAC2047 1.224 ggATgggCATATggCAAgCAg AACggAgAATTCATgCCTgCg 6 XAC3457 648 CggCATTCATATgACTCCCTT CATCTgCggATCCACATTACT Dimethyl sulfoxide 7 XAC3225 1.278 TCgggTgTCATATgATCATgC ATgCAgCCTCgAgCgTACATC 8 XAC0102 660 ATCAgCTgCggCAACAggTg AgCgggTCAgTCTgAAgACACg 9 XAC1495 405 ATATCCTCATATgTCCAAATC ATTTgACTCgAgACggATCAg 10 XAC2053 2.361 gTggTgCCTTACggTTTCAg CAgATCAgCCCATTACgACg 11 XAC3263 537 AACCACATCgCTTTCTTCCC TggATCgTTTgCTgACgg 12 XAC3285 429 ATggACTTCATgCACgACC gAACTggAAACCTggATgAgC Xcc 306

DNA samples were used in PCR performed using an initial denaturing step of 94°C for 3 min, followed by 35 cycles comprising a denaturing step of 94°C for 30 s, an annealing step at 48°C for 30 s, and a polymerization step at 72°C for 2 min. A final polymerization step of 72°C for 4 min was run, and then samples were kept at 4°C until use. The amplification reaction was carried out with 0.2 μL of DNA, 5 μL of 10× buffer, 1.0 μL of 50 mM MgCl2, 1.0 μL of 10 mM dNTP, 2.5 μL of each primer, 37.5 μL of sterile double-distilled water and 0.3 μL of Taq DNA polymerase (Invitrogen). An aliquot (5 μL) of the amplification product was electrophoresed in a 1% agarose gel, stained with ethidium bromide and visualized using an ultraviolet light transilluminator. The reaction was considered positive for a gene when the obtained product’s size was as expected. An aliquot of 400 ng of the amplified PCR product was denatured by addition of one volume of 0.