The viral isolate used in this study was WMV-M116, kindly provided by Dr. Moriones (Estación Experimental “La Mayora”-CSIC, Málaga, Spain). Mechanical inoculations were carried out using standard procedures. Dehydrated viral inoculum was first revived by mechanical inoculation of squash fully-expanded cotyledons. Systemically infected squash leaves were harvested after 15 days post-inoculation (dpi) and used as inoculum source for melons. Squash leaves were ground in a sterile mortar in the presence of inoculation buffer (0.2 M phosphate buffer pH 8.0, 0.1 % v/v β-mercaptoethanol, 0.03 g/mL active charcoal) and, then, carborundum (0.037 mm diameter) dusted 7 days old melon cotyledons were rubbed with this infectious mixture. Control non-infected melon cotyledons were rubbed using only inoculation buffer (mock-inoculated controls).
Growth protocol
Germination of melon seeds was carried out in Petri dishes for 48 h at 25 ºC. After germination, seeds were sown in 0.5 L pots and kept in growth chambers (MLR-351H from Sanyo) with 16 h light/25 ºC and 8 h dark/18 ºC for about three weeks.
Extracted molecule
total RNA
Extraction protocol
Cotyledon samples were independently frozen in liquid N2 and stored at -80 ºC. RNA extracts were prepared from these samples using Tri-Reagent (Sigma Chemical Co., St. Louis, MO, USA) following the manufacturer´s instructions. To eliminate traces of genomic DNA, total RNA was incubated with DNAse I (New England Biolabs, London, UK) 10 min at 37 ºC. Then, reaction volume was adjusted to 100 µl and the aqueous phase extracted with phenol:chloroform:isoamilalcohol (125:24:1). Total RNA was precipitated with 10 % (v/v) NaCl 3M and 2.5 v of absolute ethanol by centrifugation (12.000 g, 20 min, 4º C).
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Data processing
Data were normalised using the normalisation algorithm RMA with the package oligo version 1.8.2 (Carvalho et al. 2007, Biostatistics 8 (2): 485-499) written in R (R Development Core Team, http://www.R-project.org).
