Kemper, M. | bioRxiv Science

bioRxiv2020

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the Pospisense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

pathology↗

bioRxiv2018

Hydra Bcl-2 and TMBIMP family proteins display anti-apoptotic functions

BackgroundMechanisms of programmed cell death differ considerably between animals, plants and fungi. In animals they depend on caspases and Bcl-2 family proteins and this kind of cell death is called apoptosis. Most gene families encoding proteins involved in apoptosis are found in multicellular animals already in the eldest phyla but their functional conservation is still being studied. Much older protein families have cytoprotective functions across all kingdoms of life. This includes the TMBIMP-family, the presence and function of which in early metazoans has not been investigated yet.\n\nMethodsWe quantified apoptosis in transgenic Hydra overexpressing HyBcl-2-like 4. Moreover, we investigated putative TMBIMP-family members in Hydra by sequence comparison. By overexpression of TMBIMP-family members in Hydra and human HEK cells we analysed their subcellular localisation and in one case their capacity to protect cells from camptothecin induced apoptosis.\n\nResultsHyBcl-2-like 4, as previously shown in a heterologous system, was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis. The TMBIMP-family in Hydra includes HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4 proteins. HyBax-inhibitor-1 protein was found localised to ER-membranes, HyLifeguard-family members were found at the plasma membrane and in Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from apoptosis.\n\nConclusionThis work provides the first functional study to support an anti-apoptotic function of Bcl-2 like proteins in pre-bilaterians within a physiological context. Furthermore it illustrates that genes that were \"inherited\" from non-animal ancestors, like the TMBIMP-family, were recruited to carry out cell protective anti-apoptotic functions already in early metazoans.

developmental biology↗