Cells were treated with 50uM gomesin for 60 minutes.
Growth protocol
The virulent strain 9a5c and the avirulent strain J1a12 of Xylella fastidiosa used in this work were provided by Fundo de Defesa da Citricultura (Araraquara, São Paulo, Brazil). Both strains were isolated from Citrus sinensis (L.) Obesck trees exhibiting citrus variegated chlorosis (CVC) symptoms and established in laboratory (Li et al. 1999; Monteiro et al. 2001). Cells were reared on 2% agar periwinkle wilt (PW) broth medium (Davis et al. 1981) supplemented with 20% glucose and after seven days at 28ºC, the bacteria were transferred to liquid PW and incubated under agitation at 80 rpm for 7 additional days. The resulting pre-inoculum was diluted 1:1 in fresh PW medium and incubated at the same conditions until reaching the mid-log phase.
Extracted molecule
total RNA
Extraction protocol
A mid-log suspension of the strain 9a5c of X. fastidiosa was incubated with 50 uM of gomesin at 28ºC. As control, cells were incubated in the absence of the peptide. After 60 min, cells were harvested by centrifugation at 4.000 x g for 5 min at 25ºC and total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). To remove residual genomic DNA, samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI) and resulting RNA was quantified using a ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). In order to evaluate RNA integrity, 200 ng of each sample were electrophoresed in agarose gel, stained with ethidium bromide, and visualized using an UV transilluminator (ImageMaster VDS, GE Healthcare, Little Chalfont, England).
Label
Cy3
Label protocol
Twenty ug of total RNA (from control or gomesin-treated sample) were reverse transcribed in cDNA, which was subsequently labeled with either Alexa Fluor 555 or 647 using the SuperScript indirect cDNA labeling system (Invitrogen, Carlsbad, CA).
Cells were treated with 50uM gomesin for 60 minutes.
Growth protocol
The virulent strain 9a5c and the avirulent strain J1a12 of Xylella fastidiosa used in this work were provided by Fundo de Defesa da Citricultura (Araraquara, São Paulo, Brazil). Both strains were isolated from Citrus sinensis (L.) Obesck trees exhibiting citrus variegated chlorosis (CVC) symptoms and established in laboratory (Li et al. 1999; Monteiro et al. 2001). Cells were reared on 2% agar periwinkle wilt (PW) broth medium (Davis et al. 1981) supplemented with 20% glucose and after seven days at 28ºC, the bacteria were transferred to liquid PW and incubated under agitation at 80 rpm for 7 additional days. The resulting pre-inoculum was diluted 1:1 in fresh PW medium and incubated at the same conditions until reaching the mid-log phase.
Extracted molecule
total RNA
Extraction protocol
A mid-log suspension of the strain 9a5c of X. fastidiosa was incubated with 50 uM of gomesin at 28ºC. As control, cells were incubated in the absence of the peptide. After 60 min, cells were harvested by centrifugation at 4.000 x g for 5 min at 25ºC and total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). To remove residual genomic DNA, samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI) and resulting RNA was quantified using a ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). In order to evaluate RNA integrity, 200 ng of each sample were electrophoresed in agarose gel, stained with ethidium bromide, and visualized using an UV transilluminator (ImageMaster VDS, GE Healthcare, Little Chalfont, England).
Label
Cy5
Label protocol
Twenty ug of total RNA (from control or gomesin-treated sample) were reverse transcribed in cDNA, which was subsequently labeled with either Alexa Fluor 555 or 647 using the SuperScript indirect cDNA labeling system (Invitrogen, Carlsbad, CA).
Hybridization protocol
Fluorescent cDNAs were combined and denatured by heating to 95°C for 2 min and quickly chilled on ice. The targets were then applied to the microarray slide and covered with a 24 x 60 mm coverslip (Corning). The hybridization proceeded for 16 h at 42°C. The slides were then washed in 1X SSC + 0.2% SDS for 10 min at 55°C, twice in 0.1X SSC + 0.2% SDS also for 10 min at 55°C, 0.1X SSC for 1 min at room temperature and finally in deionized water for 1 min at room temperature and dried with N2 gas.
Scan protocol
Scanning of microarray slides was carried out using a Generation III DNA scanner (GE Healthcare, Little Chalfont, England).
Description
Biological replicate 3, technical replicate 2
Data processing
Values of Alexa Fluor 555 and Alexa Fluor 647 fluorescence intensity of each microarray spot were extracted using the Array-Vision software, version 6.0 (Imaging Research, Inc., St Catherine's, Canada). Data normalization was performed using the LOWESS method, as detailed by (Koide et al. 2004). A gene was considered differentially expressed when more than 66.5% of the biological replicates were outside the intensity-dependent cutoff curves obtained by self-self hybridization experiments (Vencio and Koide 2005) and exhibited a fold-change higher then 2.0.
