RNeasy Mini kit following the manufac turers protocol for plant tissues. cDNA AFLP analysis We used the cDNA AFLP protocol described by Vos et al. and Bachem et al. with the modifications and primers described by Breyne et al. which per selleck Olaparib mit the visualization of a single cDNA fragment for each mRNA present in the original sample, reducing the output sequence redundancy. Double stranded cDNA was synthesized from 2. 5 ug total RNA using the Super script II reverse transcription kit and a bio tinylated oligo dT primer. After pre amplification, the mixture was diluted 600 fold and 5 ul was used for selective amplification with 128 primer combinations, carried out with one selective nucleotide added on the 33P labeled BstYI primer and two selective nucleotides on the MseI primer.

We used the following touch down PCR conditions, 2 min dena turation at 94 C followed by 30 s denaturation at 94 C, 30 s annealing at 65 C, 60 s extension at 72 C for 13 cycles, 30 s denaturation at 94 C, 30 s annealing at 56 C, 60 s extension at 72 C for 23 cycles, and 5 min at 72 C. Selective ATP labeled amplification products were separated on a 6% polyacrylamide gel in a Sequi Gen GT Sequencing Cell running for 2. 5 h at 115 W and 50 C. Gels were dried onto 3 MM Whatman paper for 2 h at 80 C on a Gel Dryer Model 583 and marked with Glogos II Autorad Markers before exposing to Kodak Biomax MR films, for 12 16 h.

Sequence analysis of cDNA AFLP fragments Bands corresponding to differentially expressed genes were excised from the gels with a surgical blade and the eluted DNA was reamplified using non labeled primers identical to those employed for selective amplification and the following PCR conditions, 15 min denaturation at 94 C, 40 s denaturation at 94 C, 60 s annealing at 56 C, 40 s extension at 72 C for 35 cycles, and 5 min at 72 C. The quantity of each reamplified bands were checked on a 1. 8% agarose gel against the 1650 bp fragment of the DNA ladder 1 Kb plus. PCR products were purified with MultiScreen PCR u96 plates and sequenced directly. Sequence information was obtained by comparing nucleotide and protein sequences in the available public databases by BLAST sequence alignment. Homol ogy searching was carried out against the following data bases, NCBI, Cucurbit Genomic Database Melon Unigene ver. 4. 0, AV-951 UNIPROT database and Fusarium Comparative Database.

Sequences were manually assigned to functional categories based on the analysis of scientific literature and also with the aid of the information reported for each sequences by the Gene Ontology Consortium, where available. Sequence data from moreover this article have been deposited in GenBank, Accession Numbers, HO867279 HO867981. Real time RT PCR analysis Real time RT PCR was carried out on pools of RNA derived from two independent biological experiments. All samples were examined as three technical repli cates. Samples were prepared from whole stems of infected and mock inoculated plants correspond ing, for each gene te