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Building and bypassing plant polyspermy blocks

Periodic Reporting for period 3 - bi-BLOCK (Building and bypassing plant polyspermy blocks)

Reporting period: 2018-09-01 to 2020-02-29

"We naturally associate the term ""parents"" with two individuals, one male and one female. Work funded in this ERC project now shows that this bi-organismic concept must be expanded when it comes to plants. Our laboratory has identified genuine triparental plants, which contain the nuclear genome of one mother and two fathers.
Many organisms produce tremendous amounts of sperm to maximize the likelihood of an egg becoming fertilized. High sperm to egg ratios are, however, associated with an increased risk of supernumerary sperm fusion. This so-called polyspermy is lethal in many organisms, including humans. Accordingly, polyspermy barriers have evolved at different levels in the reproductive process. Due to the fatal outcome of supernumerary sperm fusion, the molecular nature of these checkpoints is not well understood.
We have established a polyspermy detection assay (HIPOD), which allows tracing the rare event of polyspermy in flowering plants. HIPOD revealed that supernumerary sperm fusion with one egg cell does occur in plants and can generate viable offspring. Moreover, polyspermy can give rise to seedlings with one mother and two fathers (Nakel et al. 2017).
The results have important implications for agriculture, as three-parent crosses provide a novel tool for plant hybridization. In addition, the findings shed new light on the evolution of flowering plants: The increase in genomic copies is considered a major driving force for plant evolution and our novel results now suggest that polyspermy has contributed to the amazing flowering plant diversity that shapes our planet.
Capitalizing on these unprecedented results and the newly established HIPOD assay, we will further characterize the molecular mechanisms that restrict polyspermy and analyze the consequences if these mechanisms fail.

To investigate the rare event of polyspermy, we disconnected the two gene elements necessary to engineer herbicide resistance and introduced them separately into two different plants. These plants served as fathers and were used to pollinate a third, mother plant. In case of a regular fertilization event involving one sperm from a single father only, an incomplete copy of the gene is inherited, rendering the offspring herbicide sensitive. By contrast, polyspermy, involving sperm from both fathers restores gene function and triparental plants can be identified by herbicide treatment.
We subjected more than 120 000 wild-type plants to this HIPOD assay and recovered 7 herbicide resistant polyploid plants. While these genuine triparental plants exhibit reduced fertility, they grow taller and produce bigger organs than plants resulting from a regular biparental fertilization event. Capitalizing on HIPOD we, in addition, were able to combine three Arabidopsis accessions in a single cross, thereby highlighting the hybridization potential of polyspermy (Nakel et al. 2017).
The project will identify causes and consequences of polyspermy.This study is also likely to open up new horizons for animal polyspermy research, where comparable approaches are hampered by the lethal consequences of polyspermy.