2.1 Generation of transgenic Arabidopsis
Cloning of SS1G_01703 into Agrobacterium tumefaciens followed the Gateway cloning protocol (Invitrogen, Carlsbad, CA, US) using primers found in Dataset S3 and following the methods of [8]. Target gene sequences were amplified using Phusion Taq (Thermo Scientific, Waltham, MA, US) under the following conditions: 98°C for 30 s; 35 cycles of: 98°C for 10s, 57°C for 30s, and 72°C for 30s; and a final extension of 72°C for 5 min. Amplicons were gel purified (New England Biolabs, Ipswich, MA, US) and digested using FastDigest KpnI and XhoI (Thermo Scientific, Waltham, MA, US) according to the manufacturer’s protocols. Initially, gene fragments were ligated into the pENTR4 vector before insertion into the pHellsgate8 destination vector. To confirm insert identity, entry vectors were sequenced at the Centre for Applied Genomics in Toronto, Ontario. Inserts were then recombined into the destination vector using a Gateway LR clonase reaction (ThermoFisher Scientific) following the manufacturer’s instructions, with the modification of adding 4:1 entry vector to destination vector ratio according to Wytinck et al.[8]. A cold shock treatment was used to transform Agrobacterium. Successful transformants were selected using colony PCR with XhoI and XbaI separate restriction enzyme digestions. Cultures of Agrobacterium were grown to OD 1.6-2.0, pelleted using centrifugation, and re-suspended in MS media and Silwet L-77. To transform A. thaliana, seeds were initially sterilized using alternating washes of 70% and 95% ethanol then placed on Murashige and Skoog (MS) media (Sigma Aldrich). Seeds were vernalized for 3 days at 4°C prior to being grown in a constant light incubator. Upon formation of first leaves A. thaliana seedlings were transplanted into Sunshine Mix #1 and grown to maturity at 22°C. Mature flowering A. thaliana plants were dipped into the Agrobacterium culture and kept in high humidity conditions for 2 days. Floral dips were repeated five times before seeds were dried and harvested. Transformants were selected using MS and 150 µg/mL kanamycin media [52]. PCR was used to confirm the presence of the S. Sclerotiorum gene using the same primers used for cloning within the plant (Dataset S3). Three confirmed independently transformed A. thaliana lines were grown to the T2 generation and tested for their ability to silence ABHYDROLASE-3 transcripts and slow S. sclerotiorum infection. The top performing line (AT1703.1) was subsequently selected for further RNA sequencing analysis.
2.2 Arabidopsis thaliana infection assays
Sclerotinia sclerotiorum ascospores were collected at the Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB, Canada and stored at 4˚C in desiccant in the dark according to Mcloughlin et al., 2018. S. sclerotiorum ascospore inoculum was made by suspending a 1x106 spore/mL concentration of spores in a PDB and peptone solution. Using a pipette, 10 µL of the solution was transferred onto mature transgenic and wild-type Col-0 A. thaliana leaves at a n = 15 leaves per treatment. Plants were stored in growth chambers at room temperature (21.0˚C) in humidity chambers for three days, allowing for infection progression in planta. At two- and three-days post inoculation (dpi), lesion area was quantified for n = 15 leaves per line using ImageJ software and leaves were collected for RNA sequencing, fungal load, and transcript abundance experiments. Fifteen infection sites were quantified per treatment while 3 leaves were collected per bio-replicate with three biological replicates collected per treatment. Botrytis cinerea infections were performed using ascospores from the Saint-Jean sur Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, QC, Canada following the same protocol for S. sclerotiorum ascospore inoculations.
2.3 Dna Extraction And Plant Genotyping/p>
Two-week-old A. thaliana leaves were ground using a GenoGrinder 2000 (Spex CertiPrep, Metuchen, New Jersey, USA). DNA was extracted according to Wytinck et al.[8] with DNA extraction buffer [0.25M NaCl,, 1M 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl pH 7.5, 25mM EDTA pH 8, 0.5% SDS]. DNA was precipitated with isopropanol and subsequently washed with 75% ethanol before being suspended in water. PCR was performed using GoTaq® Green Master Mix (Promega, Madison WI, USA) according to manufacturer’s instructions using ABHYDROLASE-3 specific primers (Dataset S3). Thermocycler conditions were 94°C for 2 min. followed by 35 cycles of: 94°C for 1 min., 56°C for 30s., 72°C for 35s. and a final extension at 72°C for 5 minutes. PCR products were then loaded on a 1% agarose gel containing ethidium bromide with 2 µL of sample loaded per well against FastRuler Middle Range DNA Ladder (Thermo Fisher) as a reference. Gels were visualized under UV light using the Axygen® Gel Documentation System (Axygen, Corning, NY, US).
2.4 Lactophenol cotton blue stain microscopy
A. thaliana leaf tissue was embedded in historesin (Leica, Wetzlar, GER) and vacuum infiltrated in 2.5% glutaraldehyde and 1.6% paraformaldehyde fixative solution. Methylcellosolve (Sigma-Aldrich, St. Louis, MO, USA) was used to remove pigment followed by three transfers of 100% ethanol every 24 hours to dehydrate tissue. Tissue was then infiltrated with historesin during a three-day period of increasing concentrations of resin. Day 1: 1:3 historesin:100% alcohol, day 2: 2:3 historesin:100% alcohol, and day 3: 100% historesin. Tissue was then embedded in blocks with an embedding solution of activated historesin, hardener and polyethylene glycol according to Wytinck et al. [8]. Sections were cut at 5 µM with a Leica RM2245 microtome and S. sclerotiorum infected tissues stained with lactophenol cotton blue for 20 minutes, while uninfected 0 days post inoculated tissues were stained with 0.1% toluidine blue O for 15 minutes. Uninfected and infected wild-type Col-0 and AT1703 A. thaliana leaves were imaged using a Leica DFC450C camera.
2.5 qPCR analysis
S. sclerotiorum and B. cinerea qPCR primers were designed using Primer3 (http://bioinfo.ut.ee/primer3/) and subsequently validated using the Primer BLAST tool (www.ncbi.nlm.nih.gov/tools/primer-blast). Transcript abundance was measured on the Bio-Rad CFX96 Connect Real-Time system using SsoFast EvaGreen Supermix (Bio-rad Laboratories, Hercules, CA, US) in 10 µl reactions according to manufacturer’s protocol using the following conditions: 95°C for 30s, and 45 cycles of: 95°C for 2s and 60°C for 5s. Melt curves were performed at a range of 65–95°C with 0.5°C increments to assess nonspecific amplification and primer dimers. Relative transcript accumulation was calculated using the ΔΔCt method, relative to Sac7 (SS1G_12350) for S. sclerotiorum and tubA (BC1G_00122) for B. cinerea using three biological replicates per treatment and three technical replicates per biological replicate. The same ΔΔCt method was used to quantify expression levels of RNAi machinery in S. sclerotiorum and have been used and validated in [7, 8]. All qPCR primers used in this study can be found in Dataset S3.
2.6 mRNA library preparation and RNA sequencing
RNA was isolated using the Ambion® RNaqueous® micro kit according to manufacturer’s instructions. RNA quality was analyzed using the Agilent 6000 Pico LabChip® and Agilent 2100 Bioanalyzer software (Agilent Technologies, USA). RNA quantity was determined using the Quant-iT™ RiboGreen® kit according to manufacturer’s instructions. cDNA library synthesis was performed according to Ziegler et al. [53] with three biological replicates per treatment each containing three A. thaliana leaves. Subsequently, 100 bp paired-end RNA sequencing was performed on the Illumina HiSeq4000 platform (Génome Québec Innovation Centre, McGill University, Montreal, Canada).
2.7 Rna Sequencing Analysis
Raw sequence reads were deposited at the Gene Expression Omnibus under the accession GSE217513. Read quality control and adapter sequence removal was performed using Trimmomatic 0.36 [54] (HEADCROP:9 LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 MINLEN:50). Surviving reads were aligned to the TAIR10 A. thaliana genome using HISAT2 [55] alignment software. Raw count data was generated using featureCounts [56] and inputted into pvclust (https://cran.r-project.org/web/packages/pvclust/index.html) for hierarchical clustering analysis with a detection cutoff ≥ 1. Detected transcripts were subsequently sorted into low (1 ≥ 5), moderate (5 > 25), or high (≥ 25) abundance levels. Further, raw count data was processed in DESeq2 for differential expression analysis. Here, DEGs were identified between pairwise comparisons at a p-value cutoff of p < 0.0001. GO enrichment of identified DEGs was performed using SeqEnrich [57] with terms being considered significantly enriched at P ≤ 0.001. GO heatmap visualization was performed using the conditional formatting function in Excel. GO summary tables can be found in Dataset S1. Predictive transcription factor networks were generated using SeqEnrich (Dataset S5) with DEG gene lists being used as input. Networks were subsequently visualized using Cytoscape 3.9.1. software (https://cytoscape.org/).