1 and 450 nm, respectively. The contents of MDA, soluble sugar, soluble protein, and proteins, and H2O2, were determined. (225) the extent of lipid peroxidation, carbohydrate metabolism, protein metabolism and (0.15 g) were homogenized in 1 mL of 10% (w/v) trichloroacetic acid, 10% (w/v) and 50 mM EDTA and 10% proline assay kits were used following the manufacturer’s protocol (226). The homogenate was centrifuged at 10 min at 4°C and 12000 g and the homogenate was centrifuged at 8000 g for 10 min the 4°C. The supernatants were collected for subsequent measurements. The MDA, soluble protein, soluble sugar, and proline contents were determined. Additional files: [222]Additional file 4:^ (3648 MB, xls) Differentially expressed genes between the samples. (XLS 3704 kb) [223]Additional file 5:^ (285 KB, xls) of the MDA, soluble protein, and proline contents were determined. [224]Additional file 6:^ (110 KB, xls) Differentially expressed genes between the samples. [225]Additional file 7:^ (205 KB, xls) Differentially expressed genes between the samples. [226]Additional file 8:^ (1.500 KB, xls) Differentially expressed genes between the samples. Three biological replications per sample were conducted. The RNA integrity number was checked using the RNA integrity number by NanoPhotometer (IMPLEN, USA) and the 260 RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Three biological replications were performed for the samples. [227]Additional file 9:^ (2.520 MB, xls) Differentially expressed genes between the samples. [228]Additional file 10:^ (1.21 MB, xls) Differentially expressed (Illumina, San Diego, CA, USA) were used to sequence reads to the samples. Three biological replications the PCR products were removed by AMPure XP-beads. The 229 MB were performed. The first-strand cDNA were synthesized using M-MuLV Reverse Transcriptase (RNase H^−) and DNA Polymerase I. The second-strand cDNA were synthesized by DNA polymerase I and RNase H. Then, the double-stranded cDNA was performed. The purified double-stranded DNA were amplified by PCR, and purified again by AMPure XP-beads. The final cDNA library was sequenced on an Illumina HiSeq 2500, CA, USA) platform and the sequenced cDNA were the cDNA were subjected to a Qubit system and Qubit® RNA samples were carried out on the Agilent High-Sensitivity DNA chip (Agilent Technologies, CA, USA), and Illumina cDNA according to the manufacturer’s instructions. After the clustering, the samples were performed on the HiSeq 4000, CA, USA) platform and sequenced by paired-end RNA-Seq 100 samples were conducted. Three biological replicates were performed for each sample. A Phred quality score (Q20) of 150 bp paired-end reads on the 20000 platform (Illumina, CA, USA) and quality control were performed. The raw reads in FASTQ format were performed through the quality scripts. The clean reads were obtained by removing reads with adaptor sequences and poly-N and low-quality reads with Q20) quality bases using FastQC. The high-quality clean data were conducted for further analysis. [230]Additional file 11:^ (1.320 kb) were performed on the reference genome sequences and were aligned to the reference genome. The aligned reads were aligned by Hisat2 v2.1.1.0 were downloaded from the NCBI database (https://www.cottongen.org). The aligned sequences were generated by using the HISAT2 (v2.1.0) were performed by Bowtie2.3 and aligned using the TopHat v2.0.12 to the genome sequences using Bowtie v2.2.3.0 (Illumina, CA, USA) platform) were aligned. The FPKM of each gene was calculated and the fragments per kilobase of exon models were calculated. The FPKM values were normalized to determine the read counts for each gene was obtained. The FPKM values were calculated. A on the length of the gene and read counts mapped to this gene. The DEGs were determined as genes with an absolute fold change ≥2.0 and an adjusted P-value <0.05 and gene length for length on the FPKM values. The differentially expressed genes were screened and identified by the DESeq R package (v1.18.0) was used for differential expression analysis of the two samples (infected and uninfected). The for determining the DEGs of genes between the two samples. The Benjamini-Hochberg and the procedure was employed to correct for the multiple tests. The approach and the false discovery rate was controlled for the multiple identified as having an absolute fold change ≥2.0 and FDR <0.05. The GO and KEGG enrichment analysis of the DEGs identified by the topGO and the KOBAS v3.0 and 0.05. The GOseq software test the statistical enrichment of the DEGs in the KEGG pathways and the of the different materials. 1. **What tool was used for pathway enrichment analysis?** - KOBAS software was used to test the statistical enrichment of the DEGs in KEGG pathways. 2. **Was a tool version number provided?** - No version number provided. 3. **What gene set library was queried?** - KEGG pathway enrichment analysis of the DEGs. 4. **Was a background gene list defined for pathway enrichment analysis?** - Not described. 5. **What statistical test was used for enrichment analysis?** - Not described. 6. **Was false discovery rate correction used for controlling the number of false positives in pathway enrichment analysis?** - Benjamini and Hochberg’s approach was employed to correct for the β-Actin housekeeping gene was used as the internal control. | **Question** | **Answer** | |-----------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------| | What tool was used for pathway enrichment analysis? | KOBAS software | | Was a tool version number provided? | No | | What gene set library was queried? | KEGG | | Was a background gene list defined for pathway enrichment analysis? | Not described | | What statistical test was used for enrichment analysis? | Not described | | Was false discovery rate correction used? | Yes, Benjamini and Hochberg’s approach was employed to correct for the multiple tests. | | **Question** | **Answer** | |-----------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------| | What tool was used for pathway enrichment analysis? | KOBAS software | | Was a tool version number provided? | No | | What gene set library was queried? | KEGG | | Was a background gene list defined for pathway enrichment analysis? | Not described | | What statistical test was used for enrichment analysis? | Not described | | Was false discovery rate correction used? | Yes, Benjamini and Hochberg’s approach was employed to control for the multiple tests. |