SRA

Botrytis cinerea (gray mold) is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protec-tion and is essential for controlling tomato gray mold. The emergence of fungicide-resistant strains has made the control of Botrytis cinerea more difficult. While the genome of Botrytis cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such flu-dioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) li-braries for three Botrytis cinerea strains [two highly resistant (LR and FR) versus one highly sen-sitive (S) to fludioxonil], with and without fludioxonil treatment, to identify fludioxonil responsive genes that facilitate fungicide resistance. Functional enrichment analysis identified nine resistant related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up regulated, and seven resistant related DEGs down regulated. These included adenosine tri-phosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator super-family (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxo-nil-responsive genes, obtained from the RNA-sequence data sets were validated using quantita-tive real-time PCR (qRT-PCR). Based on RNA-sequence analysis it was found that fugal HHKs, like BOS1, BcHHK2, and Bchhk17, were in some way involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8 were differen-tially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes played a crucial role in the fludioxonil resistant mechanism of B. cinerea. These lines of evidence together allowed us to draw a general portrait of the anti-fludioxonil mechanisms for Botrytis cinerea, and the assembled and annotated transcrip-tome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil. Overall design: Mycelium of the parent fludioxonil-sensitive strain (S), laboratory fludioxo-nil-resistant strain (LR), and field fludioxonil-resistant strains were obtained as de-scribed above. All of the isolates were grown at the same time for the same amount of time (three days) under the same conditions (shaking at 23°C and 180 rpm) in PDB. The mycelium were then collected at the same time. RNA was extracted from samples using the TRIzol method (Invitro Corp., Carlsbad, CA, USA).The RNA concentration and quality were assessed using a nanodrop and gelelectrophoresis. The samples were stored at ?80°C. RNA sequencing was conducted by Novogene using an Illumina? (San Diego, CA, USA) based method to generate 20 million 150-bp paired-end reads per sample. The sequencing library was prepared with the NEBNext? Ultra?RNA Library Prep Kit for Illumina?, and sequencing was performed on the NovaSeq PE150.