Computational protocol: Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression

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Protocol publication

[…] The nucleic acid sequence of Arabidopsis NPR1 was used to search an EST database of abiotically stressed leaves of V. vinifera cv Chardonnay (EST Analysis Pipeline, ESTAP [], using BLASTN. Two EST with significant similarity to AtNPR1 were identified. Full-length cDNA were reconstituted by searching the Genoscope database of grapevine genome sequencing with the two EST previously identified []. Full-length cDNAs of VvNPR1.1 (GSVIVT00016536001) and VvNPR1.2 (GSVIVT00031933001) were amplified from reverse-transcribed RNA from SA-treated Chardonnay leaves using Pfx DNA polymerase (Invitrogen). AtNPR1 cDNA was amplified from reverse-transcribed cDNA from Arabidopsis thaliana Col-0 leaves.For subcellular localization, the AtNPR1, VvNPR1.1 and VvNPR1.2 coding sequences were cloned by Gateway (Invitrogen) recombination reactions into the pK7FWG2 vector [], upstream of eGFP.For transient expression in N. benthamiana and grapevine leaves, full-length AtNPR1, VvNPR1.1 and VvNPR1.2 cDNAs were cloned between the CaMV 35S promoter and the 35S terminator sequences of the pUCAP-intron vector []. This vector contains an intron between the promoter and the terminator sequence, which was excised and replaced by NPR1 cDNA sequences. A six histidine tag coding region was added to the 3' end of each cDNA in order to facilitate detection of transgene product. The cassette containing AtNPR1, VvNPR1.1 and VvNPR1.2 between the CaMV 35S promoter and the 35S terminator was excised by AscI/PacI digestion and cloned into the pBINplus vector []. [...] Protein sequence alignment was realized using the ClustalW program. The phylogenetic tree was constructed with the Phylo_win program [], using the Neighbor-Joining method. Boostrap values were obtained from 500 replicates. […]

Pipeline specifications

Software tools ESTAP, BLASTN, Clustal W
Databases Full Length cDNA
Applications Phylogenetics, Transcription analysis
Organisms Vitis vinifera, Arabidopsis thaliana
Diseases Mycoses
Chemicals Salicylic Acid