GSE160971
Transcriptome analysis of Clavibacter michiganensis subsp. michiganensis infected tomato seedlings
The gram- positive bacterial pathogen Clavibacter michiganensis subsp. michiganensis (Cmm) causes huge economic losses by infecting tomato plants worldwide. Cmm can be spread by contaminated seeds and transplants, penetrating the plant through natural openings or wounds and is transferred through the plant xylem. While in recent years significant progress has been made to elucidate plant responses to pathogenic gram-negative bacteria by gene expression studies, the molecular mechanisms that lead to disease symptoms caused by gram-positive bacteria like Cmm remain elusive. An indigenous virulent Cmm strain isolated from a farm crop of Pomodoro tomatoes in southern Greece was used for the infection of EKSTASIS F1 hybrid tomato seedlings.  Here, we present the results of a deep RNA- sequencing (RNA-seq) analysis performed to characterize the dynamic expression profile of tomato genes upon Cmm infection. Overall design: Cmm bacteria were grown for 36hrs at 26oC in a rotating shaker, in YPDNM medium containing 1% Yeast extract, 2% Peptone, 2% D-Glucose, 1% NaCl and 10mM MgCl2. Bacteria were pelleted by centrifugation at 1200g for 10 min, washed, and diluted to a titer of 108 cfu/mL in Dilution buffer (10 mM MgCl2, 10mM Tris-HCl pH:6.8). Bacterial suspensions or dilution buffer were injected into the stem region between the cotyledons of 2 week-old plants with a syringe fitted with a 30-gauge needle. For the transcriptome studies, 1 cm sections of the stems above the inoculation site from mock treated and Cmm infected tomato seedlings were collected at 6 and 12 days post inoculation (dpi) for RNA-Seq analysis. Total RNA was extracted using the Direct-zol RNA Miniprep (Zymo research, Irvine CA, USA) kit with an on-column DNase treatment according to the manufacturer's instructions. The quantity and integrity of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and agarose gel electrophoresis. RNA-seq libraries were generated using the TruSeq Low Input kit according to the manufacturer's instructions (Illumina). Sequencing was performed on BGISEQ-500 platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample.