Exogenous dsRNA-Induced Silencing of the Phytophthora infestans Elicitin Genes inf1 and inf4 Suppresses Its Pathogenicity on Potato Plants.

Phytophthora infestans, an Oomycete pathogen, has a devastating impact on potato agriculture, leading to the extensive use of chemical fungicides to prevent its outbreaks. Spraying double-stranded RNAs to suppress specific genes of the pathogen via the RNA interference (RNAi) pathway may provide an environmentally friendly alternative to chemicals. However, this novel approach will require various target genes and application strategies to be tested. Using the L4440 backbone, we have designed two plasmids to express dsRNA targeting inf1 and inf4 genes of P. infestans that are known to contribute to the disease development at different stages. The dsRNA produced by the bacteria was tested on potato explants and demonstrated a statistically significant reduction in lesions five days after inoculation compared to water treatment. The study results allow us to consider our approach to be promising for potato late blight control.

How Does the Sample Preparation of Phytophthora infestans Mycelium Affect the Quality of Isolated RNA?

RNA isolation from fungi and fungus-like organisms is not an easy task. Active endogenous RNases quickly hydrolyze RNA after the sample collection, and the thick cell wall prevents inhibitors from penetrating the cells. Therefore, the initial collection and grinding steps may be crucial for the total RNA isolation from the mycelium. When isolating RNA from Phytophthora infestans, we varied the grinding time of the Tissue Lyser and used TRIzol and beta-mercaptoethanol to inhibit the RNase. In addition, we tested the mortar and pestle grinding of mycelium in liquid nitrogen, with this method showing the most consistent results. During the sample grinding with the Tissue Lyser device, adding an RNase inhibitor proved to be a prerequisite, and the best results were achieved using TRIzol. We considered ten different combinations of grinding conditions and isolation methods. The classical combination of a mortar and pestle, followed by TRIzol, has proved to be the most efficient.

Method for the Isolation of "RNA-seq-Quality" RNA from Human Intervertebral Discs after Mortar and Pestle Homogenization.

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells.

Investigation of cytotoxic and antioxidative activity of 1,2,3-triazolyl-modified furocoumarins and 2,3-dihydrofurocoumarins.

High biological activity of natural furocoumarins is often linked to a series of adverse side effects, e.g., genotoxicity. This makes it desirable to develop semi-synthetic derivatives with reduced negative activity while retaining or even enhancing the positive properties. Previously, we have studied the genotoxic activity of a library of twenty-one 1,2,3-triazolyl-modified furocoumarins and 2,3-dihydrofurocoumarins and identified modifications that minimize the negative properties. In the current article, we report on an investigation into the cytotoxic activity of the same library. We have aimed to rank the substances in order of the severity of their cytotoxicity and therefore to predict, with the use of statistical processing, the most promising substituents for the furocoumarin scaffold.

Phytophthora infestans: An Overview of Methods and Attempts to Combat Late Blight.

Phytophthora infestans (Mont.) de Bary is one of the main pathogens in the agricultural sector. The most affected are the Solanaceae species, with the potato (Solanum tuberosum) and the tomato (Solanum lycopersicum) being of great agricultural importance. Ornamental Solanaceae can also host the pests Petunia spp., Calibrachoa spp., as well as the wild species Solanum dulcamara, Solanum sarrachoides, etc. Annual crop losses caused by this pathogen are highly significant. Although the interaction between P. infestans and the potato has been investigated for a long time, further studies are still needed. This review summarises the basic approaches in the fight against the late blight over the past 20 years and includes four sections devoted to methods of control: (1) fungicides; (2) R-gene-based resistance of potato species; (3) RNA interference approaches; (4) other approaches to control P. infestans. Based on the latest advances, we have provided a description of the significant advantages and disadvantages of each approach.