Tomato prf3 and tft3 mutants response to Pseudomonas syringae pv. tomato (Pst) DC3000

Comparative transcriptomics between prf3, Prf-SBP-FLAG complemented lines as controls and tft3 2-2 line (CRISPR/Cas9 tomato mutant line in Rio Grande-prf3 Prf-SBP-FLAG complemented background), treated with either buffer (as control) or P. syringae DC3000 6 hours post infiltration. Leaves of four-week-old tomato prf3, Prf-SBP-FLAG and tft3 2-2 lines leaves were vacuum infiltrated with P. syringae DC3000 (OD=0.1) for 6 h; 10 mM MgCl2 infiltration served as control. All infiltrations were carried out in biological triplicate and three technical replicates were used for each. After tissue harvesting and grinding in liquid nitrogen, RNA extraction was performed using a RNeasy kit (QIAGEN) and quality checked using Bioanalyzer analysis. First-strand cDNA synthesis was performed from 2 µg of total RNA using RevertAid reverse transcriptase (Invitrogen). 200 ng of RNA was used for cDNA synthesis and Cy3-labelling using the Low Input Quick Amp Labeling Kit for One-Color Microarray-Based Gene Expression Agilent analysis. 1.65 µg of linearly amplified and labelled cDNA was hybridized for 17 h at 65oC on 4 x180k format 60-mer oligonucleotide probes designed against the S. lycopersicum cv. Heinz 1706 build 2.4 (annotation 2.5) genome (Agilent design ID = 069672; GEO record GPL21602). Each array contained ~5 probes for 34,619 transcripts. Arrays were imaged using an MS200 microarray scanner with only the 480 nm laser and using the autogain feature of the NimbleScan software.