Project properties
Title Resistance by intervention: modification of tomato DNA Primase to stop virus
Group Plant Breeding, Laboratory of
Project type thesis
Credits 24-36 ECTS
Supervisor(s) Mireille van Damme and Henk Schouten
Examiner(s) Henk Schouten
Contact info mireille.vandamme@wur.nl and henk.schouten@wur.nl
Begin date 2024/10/01
End date 2025/04/01
Description Viruses are emerging threats for crops. The best way to tackle this problem is providing the crops with resistance against viruses. This can be achieved by introgression of resistance genes. Virus-resistant cultivars can be obtained by making use of dominant plant resistance (R) genes. However, resistance conferred by dominant R-genes is not durable and can be easily overcome by newly evolving viral strains and/or species. Viral genomes encode only a small number (5-14) of proteins. Therefore, viruses fully depend on host proteins encoded by susceptibility (S-)genes for their proliferation and disease development. Recently, we have found a novel S-gene that encodes for DNA Primase Large Subunit (PriL). PriL is part of the DNA Primase complex, and essential for DNA replication of plants, but also of DNA viruses. How PriL helps the virus to replicate is still unknown, but we found a melon plant that had a variant PriL gene sequence, and this plant was resistant to ToLCNDV. The DNA replication of the plant is fine, but we expect that this variation(mutation) in PriL prevents ToLCNDV DNA replication virus. To generate durable virus disease resistant plants, we want to untangle the molecular mechanism of how viruses use the plants DNA replication system.

Research questions:
o Can we use AI to predict the 3D structure of the complex and predict which protein domains are solely relevant for viral DNA replication?
o Which other proteins and factors are part of this complex?
o Can we predict which amino acids in PriL are key and can differentiate between a resistant plant and a susceptible plant?
o Are there also variant sequences of PriL from tomato that can lead to durable ToLCNDV resistance?

Materials and methods:
The activities for the student are: Investigate allelic variation of genes in the DNA Primase complex in sequenced tomatoes and identify tomato accessions with alleles that could the DNA Primase complex. ToLCNDV disease assays on tomato to identify in allelic variant sequences could lead to resistance; Make 3D models of the DNA Primase complex proteins (including variant sequences) with viral DNA and proteins using AlphaFold3. Proteomics to validate if the variant PriL proteins are still part (or not) of the viral DNA replication complex.

Season and timing:
In view of limited availability of greenhouse space the activities should be planned well ahead. The thesis takes 6 months.
Used skills DNA/RNA isolation, Molecular cloning, PCR, qPCR, protein expression, co-immunoprecipitation (proteomics), disease assays (not with Q viruses).
Requirements PBR22803, PBR23303, PBR34803, PBR30306, PBR30806