Location: Crops Pathology and Genetics Research2013 Annual Report
1a. Objectives (from AD-416):
Prove that the presence of a wood-canker pathogen in the early stage of infection of woody tissues is detectable in green tissues, to allow for convenient sampling and early detection.
1b. Approach (from AD-416):
This detection tool targets leaves, which are more convenient samples than woody tissues, to detect the early stage of infection, before symptoms appear. To identify unique molecular signatures in the leaves that correspond to the early stage of infection in the woody tissues, we combine advanced scientific techniques (High Resolution Computed Tomography; HCRT, RNA-seq) as a new research tool for canker-diseases. An early detection tool will allow nurseries to detect infected plants, in order to prevent the spread of wood-canker diseases via contaminated nursery stock. Our approach is to identify unique plant-molecular responses in green tissues, using RNA-seq, that are associated with plant-anatomical responses in woody tissues, as visualized using light/confocal microscopy and HRCT. During the funding period, the postdoc will analyze the RNA-seq data from the inoculation experiments in the greenhouse.
3. Progress Report:
This project was established in support of objective 1 of the in-house project, which is to develop sustainable disease control practices for grapevines. The goal of this project is to prove that the presence of a wood-canker pathogen in the early stage of infection of woody tissues is detectable in green tissues, to allow for convenient sampling and early detection. The overall aim of this project is to identify early transcriptomic signatures of grapevine in response to infection with the canker dieback causing agent, Neofusicoccum parvum. To achieve this goal, biological replicate experiments have been conducted in the greenhouse to mimic field conditions and to be able to replicate those experiments under nearly identical conditions. The first experiment has been carried out in the Fall of 2012 on 85 grapevines and the second experiment in Spring/Summer, 2013, on 288 vines. As the progression of the disease is complex and usually takes several months for the first symptoms to appear, we designed both replicate experiments with sample points from 0.5 months to 2 months after infection (1st experiment) and 3 hours to 3 months (2nd experiment) to include early, symptom free and late, symptom-containing samples in our subsequent analysis. Plants have been either non-infected but wounded (NIW), non-infected and non-wounded (NINW) and infected wounded (IW) to be able to differentiate the transcriptomic responses caused by common stress applied to the plants such as wounding (NIW) from transcriptomic responses caused by the pathogen of interest, Neofusicoccum parvum (IW). At indicated times from 0 hours, which was the day of inoculation of the grapevines with the pathogen, leaves have been collected, immediately frozen, ground under liquid nitrogen and aliquoted for subsequent analysis. To date (summer 2013), RNA has been extracted from all time-points of the first experiment (during spring 2013) and times 0 hours and 3 hours of the 2nd experiment. Later time-points of the 2nd experiments have been harvested and stored at -80°C or are still awaiting sampling (3 month time point at the end of August, 2013). Total RNA has been or will be extracted from pooled leaves of NIW, NINW and IW groups. To increase reliability of data and minimize biological variability of the individual plants, in the 1st experiment, leaves of 2 plants have been pooled into one group for NIW, whereas leaves of 6 plants have been pooled into one group for IW and NINW. The reliability of experimental replication has been optimized in the 2nd experiment with all groups (NIW, NINW and IW) containing samples groups consisting of 4 individual plants. During summer 2013, quality parameters of the RNA have been evaluated based on an Experion automated electrophoresis system for RNA integrity producing electropherograms showing intact ribosomal RNA bands; Nanodrop technology for RNA purity from the ratio of the absorbance at 260nm (A260nm) to the absorbance at 280nm (A280nm) and 230nm (A230nm) with ratios between 1.8 and 2.2; and, Fluorescent probe labeling using RiboGreen for RNA concentration prior to Experion analysis. This method is roughly 1000 fold more sensitive than UV spectroscopy and more reliable in the presence of common contaminants of RNA preparations (see above mentioned molecules absorbing at A230nm). However, the RiboGreen reagent interacts with all nucleic acids including RNA and DNA, therefore genomic DNA was removed using on-column digestion during the extraction of total RNAs. In addition, subsequent analysis using Next Generation Sequencing Technology (RNASeq) demands genomic DNA free RNA to avoid reads originating from DNA templates. This is important considering the fact specific non-transcribed regions of the plant genome contain polyA enriched sequences which could be selected during polyA enrichment of mRNAs within extracted RNA pools. Total RNAs from the 1st experiment have been shipped to Universtiy of California (UC), Davis, sequencing facility to produce mRNA libraries. During summer, 2013, libraries will be quality controlled using Bioanalyzer trace analysis. Quality libraries will be inserted into the HiSeq2500 system from Illumina and sequenced to produce 50 bp single-end reads from our RNA to identify transcripts differentially expressed in response to infection of grapevines with Neofusicoccum parvum by comparing the classes IW and NIW. During bioinformatical analysis of the 1st experiment and quality control of read data obtained from it (Fall 2013), RNA extraction of the 2nd experiment will be finished in preparation for sequencing during late Fall 2013.