Periodic Reporting for period 1 - NUCLAGY (NUCLEAR DYNAMICS OF AUTOPHAGY IN PLANT IMMUNITY)
Período documentado: 2020-11-01 hasta 2022-10-31
The NUCLAGY project aimed at advancing our understanding of how autophagy functions in plant immunity with the long-term goal to identify new strategies for the improvement of disease resistance in crop species. In general, autophagy is a well-studied degradation and recycling pathway in eukaryotic organisms, yet most of the knowledge in plants has accumulated on its cytoplasmic targets and mechanisms during development and stress responses. NUCLAGY therefore intended to particularly address the largely unexplored nuclear events of autophagy and to uncover the potential role of autophagic targets and activities in the nucleus during plant virus infection. Based on our previous observation that several isoforms of the ATG8 protein family have the capacity to localise to distinct subcompartments in the nucleus, it was hypothesised that ATG8 proteins (i) contribute to the autophagic degradation of nuclear substrates (nucleophagy), (ii) are involved in the nuclear regulation of pro- or antiviral autophagy activities and/or (iii) intersect with viral effector proteins. The following research objectives were addressed by the different working packages:
1. Identification of nuclear components associated with autophagy at normal and virus-induced conditions
2. Investigation of the functional relationship of isolated nuclear components with autophagy regulation or selective degradation
3. Analysis of the interplay of viruses and their effectors with the nuclear events of autophagy.
Overall, the outcome of NUCLAGY provided strong evidence for important roles of nuclear autophagy during plant virus infection and indicated that ATG8-related functions in the nucleus can contribute both to antiviral defences and viral pathogenicity. The identification of ATG8-associated proteins revealed several novel candidates as nuclear autophagic substrates or potential transcriptional/epigenetic regulators, and pointed to a distinct nuclear subcompartment as an emerging battlefield for the autophagy-pathogen interplay. These fundamental new insights hold promise for translational applications and may serve in the future breeding-based approaches to enhance crop fitness and productivity for sustainable agriculture.
1. Identification of nuclear components associated with autophagy at normal and virus-induced conditions
2. Investigation of the functional relationship of isolated nuclear components with autophagy regulation or selective degradation
3. Analysis of the interplay of viruses and their effectors with the nuclear events of autophagy.
Overall, the outcome of NUCLAGY provided strong evidence for important roles of nuclear autophagy during plant virus infection and indicated that ATG8-related functions in the nucleus can contribute both to antiviral defences and viral pathogenicity. The identification of ATG8-associated proteins revealed several novel candidates as nuclear autophagic substrates or potential transcriptional/epigenetic regulators, and pointed to a distinct nuclear subcompartment as an emerging battlefield for the autophagy-pathogen interplay. These fundamental new insights hold promise for translational applications and may serve in the future breeding-based approaches to enhance crop fitness and productivity for sustainable agriculture.
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.
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.
NUCLAGY has substantially increased our fundamental knowledge on the nuclear processes associated with autophagy in the context of plant pathogen infections. We were able to generate an inventory of the proteins associated with nuclear ATG8, providing an enormous resource for future studies on the nuclear substrates, cargo receptors and regulators of autophagy. Hence, our research proved to be timely, novel and highly original as it adds to the increasing appreciation of the nuclear roles of autophagy in yeast and mammalian systems. The discovery of the ATG8 interactome will further provide numerous potential targets for autophagy manipulation in crop species which is not only expected to benefit disease resistance to several economically important pathogens but also to improve a multitude of additional agricultural traits related to plant productivity and tolerance to abiotic stresses. Autophagy modulation by next-generating breeding technologies will therefore likely contribute to future sustainable agricultural production systems and global food security in light of the ever-growing global population and rapid climate change.
Besides the scientific progress in relation to both basic and applied research questions, the outcome of NUCLAGY will have substantial impact for the promotion of collaboration with other academic researches, stakeholders, and the industrial sector, and will also foster societal awareness and understanding of plant biology and related fields. Finally, at the personal level, the project allowed substantial knowledge transfer and provided enormous opportunities and new career paths for the researcher´s professional development in academia and/or industry. On the other side, the host laboratory and department significantly gained from the researcher´s expertise on plant and virus systems, not only in performing high-profile research but also in providing an excellent environment for education and scientific training of Master and PhD students.
Besides the scientific progress in relation to both basic and applied research questions, the outcome of NUCLAGY will have substantial impact for the promotion of collaboration with other academic researches, stakeholders, and the industrial sector, and will also foster societal awareness and understanding of plant biology and related fields. Finally, at the personal level, the project allowed substantial knowledge transfer and provided enormous opportunities and new career paths for the researcher´s professional development in academia and/or industry. On the other side, the host laboratory and department significantly gained from the researcher´s expertise on plant and virus systems, not only in performing high-profile research but also in providing an excellent environment for education and scientific training of Master and PhD students.