EXPLORATION OF PLANT EARLY RESPONSE MECHANISMS TO SELF-DNA EXPOSURE

My PhD thesis is structured into six comprehensive chapters, each aimed at exploring different aspects of the plant's early response mechanisms to self-DNA exposure. The first chapter serves as an introduction and synopsis, offering a detailed overview of the main research topics and unresolved questions. In the second chapter, we conducted a cross-factorial experiment to investigate the species-specificity of self-DNA inhibition in closely related species. By exposing Setaria pumila and Setaria italica seedlings to self-DNA, congeneric DNA, and plant and animal heterospecific DNA for four days, we confirmed a significant root growth inhibition by self-DNA, while non-self treatments exhibited effects consistent with the phylogenetic distance between the DNA source and the target species. The third chapter focuses on a Real-Time qPCR analysis of abiotic stress-responsive genes following self-DNA exposure in Setaria pumila and Setaria italica seedlings. Our targeted gene expression analysis revealed the early activation (1 to 3 hours after self-DNA exposure) of genes involved in ROS degradation and management, along with the deactivation of negative regulators of stress signaling pathways. In the fourth chapter, we explored the feasibility of a revised DNA-RNA immunoprecipitation protocol for detecting hybrid-induced formation in vitro. This revised protocol was then applied in vivo to Arabidopsis seedlings without prior self-DNA exposure to verify the absence of target gene amplification under normal conditions. Subsequently, we performed DNA-RNA immunoprecipitation on Arabidopsis seedling roots exposed to self-DNA probes targeting specific genes. Our results indicated the amplification of target genes, suggesting DNA-RNA hybrid formation, while off-target genes showed no amplification. We also tested whether the hybrid formation could derive from probe carry-over on roots during the nucleic acid extraction phase by adding the probes directly into the lysis buffer. Unfortunately, we obtained the same result produced by self-DNA probe exposure. Root treatment with DNase previous nucleic acid extraction did not provide conclusive results. In the fifth chapter, exploring epigenetic effects by self-DNA exposure, we exposed Arabidopsis thaliana seedlings to self-DNA for 6 and 24 h. For each sample, approximately 25% of the collected roots were used for RNA extraction and subsequent mRNA-seq analysis, while the remaining roots were subjected to DNA extraction for whole-genome bisulphite sequencing (WGBS). Preliminary sequencing results, analysed using bioinformatic tools, indicated potential gene expression and methylation changes in the treated genomes after 24 hours of self-DNA exposure. However, confirmatory analyses and further investigations are required to elucidate the specific genes and genomic regions involved. Finally, the sixth chapter provides conclusive remarks regarding the research questions addressed in the preceding chapters, offering valuable perspectives and guidelines for future experimental work.