The infection of grape from a pathogen induces a global transcriptional reprogramming which allows the activation of defence mechanisms. Such reprogramming network is made of transcripts differentially regulated during the infection and part of this regulation is made by conserved regulatory functions of miRNA. The aim of this Milestone is the identification and the analysis of the arrangement and distribution of coding and small non coding RNAs in infected leaves of grape selected during the first year of the project according to the results obtained in Milestone 3. In particular, individuals showing favourable (resistant to PM) or unfavourable (susceptible to PM) QTL alleles will be selected and total RNA sequencing will be used to detect and quantify the coding RNAs and the small non coding RNAs (so called microRNAs).
Activity 4.1: Selection of the genotypes to be used for trancriptomic analysis
Objective: Select a subset of samples showing opposite behaviour in terms of PM resistance
Expected results:
- Identification of the individuals characterized by high susceptibility and resistance (ER 4.1).
Methodologies:
The results of Milestone 3 will enable the identification of genomic regions associated to resistance/susceptibility to fungi. The identification of QTL regions will enable the selection in silico of candidate genes in the region(s) harbouring the QTL. Individuals showing opposite QTL genotypes (thus opposite phenotypes) will be selected for the analysis of the expressed genes and regulatory sequences through the sequencing of coding and small non coding RNAs.
Activity 4.2: Powdery mildew inoculation test on selected genotypes and RNA extraction
Objective: Evaluation of the response to powdery mildew of individuals characterized by high susceptibility and resistance
Expected results:
- Extraction of high quality RNAs for RNAseq and small RNA sequencing
Methodologies:
The in vitro assay will be carried out by using young fully developed leaves placed on 1% water-agar in plastic Petri dishes. Leaves will be infected with powdery mildew using fresh conidia collected from naturally infected leaves of a susceptible cultivar. The leaves will be placed in a climate chamber at 25 °C and 65% relative humidity on a 16-h light, 8-h dark cycle, for the development of the fungus. Leaves will be sampled in three independent pools (biological replicates) of 5 leaves at 0, 12 and 24 hours post infection (HPI), immediately frozen in liquid nitrogen, and stored at -80°C for subsequent analyses. Total RNA will be isolated from 40 mg of ground leaves. The Spectrum™ Plant Total RNA kit (Sigma-Aldrich) will be used following the manufacturer’s protocol for total RNA isolation and microRNAs recovery.
Activity 4.3: Sequencing of coding and small non-coding RNAs (sncRNA)
Objective: identification of differentially expressed mRNAs and sncRNAs in highly resistant and highly susceptible genotypes during powdery mildew infection.
Expected results:
- Provide an expression dataset of powdery mildew infected leaves
- Identification of a detailed overview of microRNAs active during infection
- Validation of the QTL analysis
Methodologies:
Total RNA from selected grapevine individuals will be used to prepare cDNA libraries for high-coverage RNA-Seq (10M reads) on an Illumina Hi-seq2500. Raw reads will be mapped to the V. vinifera genome using STAR, and DEGs will be identified with DESeq2. Transcriptomic data will help identify key pathways and genes related to PM defense. For small RNAs, 24 libraries from infected and control leaves will be sequenced. Sequences will be processed using Skewer and mapped with Bowtie. miRNAs will be identified using miR-prefer and Short-Blastn. Differentially expressed mRNAs and miRNAs will be validated by qRT-PCR, with expression quantified using the ΔΔCt method. RNA-Seq data will further validate candidate genes by comparing expression between susceptible and resistant individuals.