Among the hundreds of ribosomal RNA (rRNA) gene copies organized as tandem repeats in the nucleolus organizer regions (NORs), only a portion is usually actively expressed in the nucleolus and participate in the ribosome biogenesis process. The role of these extra-copies remains elusive, but previous studies suggested their importance in genome stability and global gene expression. Because the nucleolus is also a platform for nuclear organization, we tested the impact of a decreased amount of rRNA gene copies on the Arabidopsis thaliana 3D genome organization and stability, using an A. thaliana line only containing 20% of rRNA gene copies (20rDNA line). Compared to the wild-type Col-0, the 20rDNA line shows several signs of genomic instability, such as variations in 3D genome organization, spontaneous double-strand breaks accumulation, transcriptomic changes, and higher DNA methylation level. Strikingly, using genomic and microscopic approaches, we identified seven large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. As a consequence, more than 600 genes were duplicated, often associated with an increase in their expression level. Among them, we found several upregulated genes involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and we discuss their potential implications on plant genome evolution.