Stabilisation of <i>Tomato Yellow Leaf Curl China Virus</i> Infectious Clones Through Micro‐Homology Mediated End Joining

Tomato yellow leaf curl China virus (TYLCCNV) is a major agricultural pathogen and primary model for circular single-stranded DNA (cssDNA) virus studies. The infectious clones of TYLCCNV and other cssDNA viruses are usually constructed as two tandem copies of the small viral genomes in Agrobacterium-mediated T-DNA vectors. However, during our experiment, we observed that successive cultivation of the agrobacterial infectious clone of TYLCCNV led to a reduction or complete loss of its virulence in host plants. Further analysis revealed that the instability of this infectious clone is analogous to the mechanism of viral genome release from the dimeric infectious clones of cssDNA viruses during rolling circle replication, with key contributing factors being the activity of the viral Replication protein (Rep) and the replication origins. Unlike the infectious clones of RNA viruses, which often utilize introns to disrupt toxic protein coding sequences in order to achieve stabilization, the infectious clones of DNA viruses are unable to remove introns before releasing viral genomes. To address this challenge, we developed a micro-homology mediated end joining (MMEJ)-based system that disrupts the Rep coding sequences with an I-SceI site flanked by microhomologous regions, stabilizing the infectious clone in prokaryotic cells. Then the transiently co-expressed I-SceI enzyme seamlessly removes the introduced I-SceI site through MMEJ repair in plant hosts, resulting in efficient release of functional TYLCCNV viral genomes in planta. Theoretically, this approach can be applied to the construction of stable infectious clones for all plant DNA viruses.