To address the three main objectives of NUCLAGY, a multidisciplinary approach with a combination of genetic, cell biological, biochemical, proteomic and bioinformatic techniques has been applied. Subcellular localization studies in the plant model Arabidopsis thaliana revealed the specific targeting of an ATG8 isoform to a distinct subnuclear compartment upon challenge with Cauliflower mosaic virus, a pararetrovirus with a double-stranded DNA genome. Based on this finding, a targeted candidate as well as proteomics-based screening approach was followed to identify ATG8-associated proteins in CaMV-infected plants. Genetic intervention experiment with nuclear proteins from the ATG8 interactome revealed the contribution of an epigenetic modifier and potential transcriptional regulator to antiviral immunity, whereas other proteins involved in nuclear and genome integrity proved to be important for viral disease progression. We could also show that the viral capsid protein (P4) co-localises with ATG8 and associated proteins in the nucleus, implying the possibility that P4 targets nuclear proteins to suppress antiviral responses, which in turn might be counteracted by ATG8 to facilitate P4 degradation. Finally, we identified a kinesin-like motor protein as a highly enriched protein in the ATG8a interactome. Although we did not find strong support for a direct link to nuclear autophagy events, we discovered an exciting pro-viral role for this kinesin in CaMV infection by safeguarding viral particles from xenophagic degradation and thus, facilitating virus accumulation and transmission.
Together, these major results will be further exploited in follow-studies to strengthen the links of the candidate proteins to ATG8-related autophagy processes in immune responses as well as their potential recruitment or counteraction by viral effectors for enhanced pathogenicity. The most significant and novel findings are planned to be published open-access in scientific journals. In this context, a manuscript describing our key discoveries in relation to the kinesin-related protein is in the process of submission to a pre-print server and for peer-review at a high-profile journal (tentative title: A plant virus exploits actin filament-binding kinesins to evade xenophagy by Kushwaha et al.) In addition, we will also consider the possibility to commercially exploit our unpublished findings through patent applications in collaboration with SLU Holding. Throughout the grant period and beyond, we have communicated our findings internally through seminars and externally through conferences including e.g. the 6th Nordic Autophagy Meeting and 12th International Congress of Plant Pathology. Furthermore, the subject of NUCLAGY has been used for educational purposes, e.g. as part of a Master thesis work. Finally, the results are already used or will be further exploited as a foundation for grant applications at both national and international level.
