Impact of P. Mediterranea Pre-Treatment on Genome-Wide DNA methylation in Plenodomus tracheiphilus-Inoculated lemon (Citrus limon L.) plants

• P. mediterranea alters the methylation status of the CHH context of promoter regions. • Genes were grouped based on the opposite trends between expression and methylation. • Both hypo/hyper methylated genes are implicated in coinciding biological processes. • A negative regulator of plant immunity is a promising candidate for genome editing. Lemon ( Citrus limon L. Burm.) is widely cultivated in the Mediterranean and Black Sea regions where it is challenged by mal secco (MSD), a disruptive disease caused by the Plenodomus tracheiphilus fungus. The application of biocontrol agents (BCAs) has garnered growing interest due to the necessity of mitigating pathogen-induced yield losses while reducing the reliance on harmful substances for pathogen control. Pseudomonas mediterranea strain PVCT 3C pre-treatment has revealed a promising strategy to cope with mal secco disease with positive impacts mainly achieved by impeding the fungus-induced reprogramming of lemon leaf transcriptome. In this study, the effect of P. mediterranea PVCT 3C pre-treatment upon the DNA methylation status of fungal inoculated lemon plants was investigated via whole genome bisulfite sequencing (WGBS). The study reveals that PVCT 3C application mainly alters the methylation status of the CHH context of promoter regions. In the plants pre-treated with the biocontrol agent and P. tracheiphilus inoculated vs plants P. tracheiphilus inoculated comparison (3CPt vs Pt), a total of 1,873 differentially methylated regions (DMRs) were identified, while DESeq2 analysis of transcriptomic data detected 1,072 differentially expressed genes (DEGs). The integration of transcriptomic and methylation data allowed the retrieved genes to be grouped based on the opposite trends between expression and methylation. Most of them are implicated in coinciding biological processes, suggesting their key role in plant resilience induced by the bacterial BCA treatment. Among the hypermethylated/downregulated genes, a calcium-binding protein negatively regulating plant immunity was identified, representing a favorite candidate for genome editing application in lemon biotechnological breeding.