Elucidating the Function of Myosin XI in Polarized Cell Growth and Investigating the Chitin-induced Calcium-mediated Immune Response in P. patens.

Galotto, Giulia

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Elucidating the Function of Myosin XI in Polarized Cell Growth and Investigating the Chitin-induced Calcium-mediated Immune Response in P. patens.

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To protect and exploit existing plant species, it is advantageous to understand the molecular mechanisms behind plant growth and defense response to threats. A subgroup of essential specialized plant cells grows via a highly anisotropic fashion referred to as “tip growth”. The elongated shape of these cells emerges from polarized expansion; this process is mainly driven by myosin XI and the F-actin cytoskeleton mediated vesicle trafficking to the cell tip. Loss of function of myosin XI and F-actin cease tip growth, but the mechanism by which they self-organize to maintain growth has not been characterized. Furthermore, the role of myosin XI in vesicle trafficking in plants has been an underlying assumption in the field, but direct evidence is lacking. Cross correlation studies show myosin XI anticipate the F-actin signal at the cell tip, suggesting myosin XI could be involved in enriching the local F-actin concentration. To shed light on myosin XI involvement in F-actin concentration and vesicle transport, we generated a myosin XI temperature sensitive allele in the moss model system Physcomitrium (Physcomitrella) patens. P. patens amenability to genetic manipulation, and reduced myosin family size compared to vascular plants facilitate study of protein function. By using this tool, we demonstrated myosin XI clusters secretory vesicles, and drives a mechanism that result in F-actin polymerization. In addition, we proved myosin XI is important in maintaining cell morphology, vacuole homeostasis and cell viability. Understanding mechanisms behind plant immune response provide a great advantage to increase plant resistance to threats. In vascular plants, various aspects of the immune response against biotic and abiotic stresses have been characterized. These include cytoplasmic Ca2+ concentration increase and MAPK phosphorylation cascade that triggers synthesis of defense hormones, activation of defense related genes and lignification of the cell wall. In land plants, only the plant immune response to an abiotic stress has been object of studies, but mechanisms related to biotic stress response are still lacking. To fill this gap, we studied the response of P. patens to a fungal infection simulated by chitin. Since cytoplasmic Ca2+ concentration is the first effect observed in vascular plants, we generated a P. patens line expressing the Ca2+ reporter GCaMPf6, to investigate if a similar Ca2+ signature is present in land plants. The Ca2+ reporter line also expresses Lifeact-Ruby2, to simultaneously visualize F-actin and Ca2+. Our results show, upon chitin treatment, Ca2+ oscillation propagates across the plant in a dose-dependent manner. Furthermore, we showed chitin affects growth at the cell and plant level, via affecting the F-actin cytoskeleton at the cell tip, essential for growth. Finally, we show chitin induced Ca2+ increase triggers transcription of defense related genes.

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