Biology subjects

Minina, A. E. A.

Publications and source records attributed to Minina, A. E. A..

4 recordsLinked to original sources

bioRxiv2021

RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools. Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition. We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.

cell biology↗

bioRxiv2021

Light at the end of the tunnel: FRAP assay reveals that plant vacuoles start as a tubular network

Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study impeding reaching the consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum being the main membrane contributor for the tubular network of young vacuoles was recently challenged in a study proposing that young plant vacuoles comprise a set of individual vesicles that are formed de novo via homotypic fusion of multivesicular bodies (MVBs). To resolve these seemingly contradictory observations we have carefully revaluated both hypotheses. Here we provide a systematic overview of successive vacuolar biogenesis stages in Arabidopsis root, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelles morphology and provide 3D models of vacuoles at all developmental stages. Furthermore, we established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Finally, we summarized the new and pre-existing evidence substantiating that vacuolar structures cannot originate solely from MVBs.

plant biology↗

bioRxiv2021

Self-ubiquitination of a pathogen type-III effector traps and blocks the autophagy machinery to promote disease

Beyond its role in cellular homeostasis, autophagy plays anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One prominent role of anti-microbial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in depletion of a bacterial virulence factor and unravels an unprecedented pathogen strategy to counteract defense-related autophagy.

plant biology↗

bioRxiv2021

SPIRO-the automated Petri plate imaging platform designed by biologists, for biologists

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIROs excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared to manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing and photoblastic response.

plant biology↗