Overexpression of Full-length Expansin A4 or its N-terminal Domain is Toxic in Physcomitrium patens

Davis, Gabriella B.

Student Work

Overexpression of Full-length Expansin A4 or its N-terminal Domain is Toxic in Physcomitrium patens

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Plant expansins are a large family of cell-wall anchored proteins involved in cell wall loosening, plant growth and development, and organogenesis. The objective of this project was to identify an expansin gene from the model plant, Phycomitrium patens, that is highly expressed in tip-growing protonemata, and use it to localize the sites of active secretion. We identified PpEXPA4 as a gene highly expressed in protonemata and created constructs of PpEXPA4 tagged with either a C-terminal HaloTag or a C-terminal triple fluorescent protein. For our initial study, we expressed the proteins from a strong constitutive promoter. Our hypothesis was that PpEXPA4 would be secreted and accumulate on the cell wall. Instead, we found that high levels of expression of PpEXPA4 were toxic to the cell. To alleviate this and partially disrupt expansin’s activity, we removed the C-terminal domain of PpEXPA4 and created tagged constructs containing only the tagged domain 1 of PpEXPA4. Expression of this construct, even at lower concentrations, was also found to be toxic. Although the tagged protein was identified in the cell’s interior, none could be identified on the cell wall. Our results suggest that the toxicity of PpEXPA4 at high levels is due to domain 1 of the protein. Future studies should evaluate the localization and toxicity of PpEXPA4 expressed from its endogenous promoter as well as the overexpression of domain 2.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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