bioRxiv ScienceSearch

Biology subjects

Galotto, G.

Publications and source records attributed to Galotto, G..

2 recordsLinked to original sources

Myosin XI drives polarized growth by vesicle clustering and local enrichment of F-actin in Physcomitrium (Physcomitrella) patens

In tip-growing plant cells, growth results from myosin XI and F-actin mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cells tip, suggesting a mechanism where vesicle clustering via myosin XI promotes F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating Physcomitrium (Physcomitrella) patens plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability, but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI clusters and directs vesicles at the tip of the cell, which induces F-actin polymerization, increasing F-actins local concentration. Our findings support the role of myosin XI in vesicle clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

cell biology

Robust survival-based RNAi using in tandem silencing of adenine phosphoribosyltransferase

RNA interference (RNAi) enables flexible and dynamic interrogation of entire gene families or essential genes without the need for exogenous proteins, unlike CRISPR-Cas technology. Unfortunately, isolation of plants undergoing potent gene silencing requires laborious design, visual screening, and physical separation for downstream characterization. Here, we developed a novel APT-based RNAi technology (APTi) in Physcomitrella patens that simultaneously improves upon the multiple limitations of current RNAi techniques. APTi exploits the pro-survival output of transiently silencing the APT gene in the presence of 2-fluoradenine, thereby establishing survival itself as a reporter of RNAi. To maximize silencing efficacy of gene targets we created vectors that facilitate insertion of any gene target sequence in tandem with the APT silencing motif. The APTi approach resulted in a homogenous population of P. patens mutants specific for our gene target, with zero surviving background plants within 8 days. The observed mutants directly corresponded to a maximal 93% reduction of the tested target protein, substantially exceeding previous dsRNA methods. The positive selection nature of APTi represents a fundamental improvement in RNAi technology and will contribute to the growing demand for technologies amenable to high-throughput phenotyping. One-sentence summaryGeneration of dsRNA targeting the APT gene in tandem with a target gene enables positive selection of strongly silencing plants.

plant biology