Transcriptom analysis of two types of cell death by RNA-seq

We established an Arabidopsis thaliana Columbia (Col-0) and Cucumber mosaic virus strain HYY [CMV(HYY)] patho-system and identified a necrotic cell death. The global gene expression analysis of the necrotic cell death and HR cell death developed in cucumber mosaic virus-inoculated leaves of A. thaliana was performed by RNA-seq, respectively. The samples for RNA-seq were collected from the cell death leaves showing HR cell death at 3dpi and showing necrotic cell death at 5dpi, respectively. The sequence data was first preprocessed by FASTQ in basespace of illumine.The sequence reads were processed with Trimmomatic (version 0.38) for adaptor trimming and quality trimming. The processed reads were mapped to the genome sequences of A.thaliana ecotype Col-0, CMV(Y) and CMV(Ho) with STAR (version 2.7) with its default settings. Read counts per each A.thaliana gene were retrieved by the quantification option of STAR. The obtained read counts were normalized and statistically tested using DESeq2 R package 3.7. Adjusted p-values were calculated with Benjamini-Hochberg procedure, and the threshold for adjusted p-value was set to 0.05 in the present study. The independent filtering in DESeq2 was performed to filter out the genes with low mean normalized counts, with an automatically optimized threshold; 5,906 genes which passed the independent filtering in both of the two comparisons (i.e, necrotic cell death vs mock and HR cell death vs control) were further analyzed for differential expression. Genes with adjusted p-value of <0.05 and a fold change of >4 or <0.25 were considered as DEGs. Of the 5,906 genes analyzed, 202 genes showed >4-fold up-regulation upon either HR cell death or necrotic cell death induction (adjusted p < 0.05). Thirty-five of them were common in HR cell death or necrotic cell death induction, while 149 and 18 were unique to HR cell death or necrotic cell death induction, respectively. Sixty-two genes showed <0.25-fold down-regulation upon either HR or necrosis induction (adjusted p < 0.05). Seven of them were common , while 43 and 12 were specific for HR cell death or necrotic cell death induction, respectively. Collectively, the necrotic cell death displayed a distinct gene expression pattern compare to HR cell death. Overall design: Transcriptome analysis of "Necrosis" and HR cell death developed in cucumber mosaic virus-inoculated leaves of Arabidopsis thaliana was performed by RNA-seq, in triplicate, using TruSeq Stranded Total RNA Sample Preparation Kit.