Transcriptome profiling and statistical analysis
To elucidate the putative transcriptional function of Pik-H4 , transcriptome profiling was performed by RNA-seq. For pathways analysis post-inoculation, three- to four-week-old LTH and Pik-H4 NILs seedlings were spray inoculated with M. oryzae isolate GDYJ7 as previously described. Up to 0.1 g shoots were sampled at 0 (as mock treatment), 12 and 24 hpi. The inoculation assay was estimated by LTH phenotype 10 to 14 dpi. For singleton Pik-H4 pathways analysis, three- to four-week-old OE-Pik1-H4 -GFP /Pik , OE-Pik2-H4 -GFP /Pik andPik-H4 NILs shoots were sampled. Total RNA was extracted using a Trizol reagent kit (Invitrogen) and the mRNA was enriched by the Oligo(dT) beads. Then, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversely transcribed into cDNA using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). The purified double-stranded cDNA fragments were end-repaired, A base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads(1.0X). And polymerase chain reaction (PCR) amplified. The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Differentially expressed genes (DEGs) were identified withP <0.01, |log2Foldchange|≥2. Correlation analysis was performed by R. Correlation of two parallel experiments provides the evaluation of the reliability of experimental results and operational stability. The correlation coefficient between two replicas was calculated to evaluate repeatability between samples. GO enrichment analysis was performed using TBtools (v1.120).