A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference

Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named “ASP-score”. The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.     

SlAGO4A,a core factor of RNA-directed DNA methylation (RdDM) pathway,plays an important role under salt and drought stress in tomato

In Arabidopsis, it has been clarified that AGO4 protein is implicated in a phenomenon termed RNA-directed DNA methylation (RdDM). Previously, four orthologs of AtAGO4 were cloned in tomato, designated as SlAGO4A–SlAGO4D. Here, we studied the role of the SlAGO4A gene in regulating salt and drought tolerance in tomato. SlAGO4A-down-regulating (AS) transgenic tomato plants showed enhanced tolerance to salt and drought stress compared to wild-type (WT) and SlAGO4A-overexpressing (OE) transgenic plants, as assessed by physiological parameters such as seed germination rate, primary root length, chlorophyll/proline/MDA/soluble sugar/RWC content, and survival rate. Moreover, several genes involved in ROS scavenging and plant defense, including CAT, SOD, GST, POD, APX, LOX, and PR1, were up- or down-regulated consistently under salt and drought stress. Notably, expression levels of some DNA methyltransferase genes and RNAi pathway genes were significantly lower in AS plants than in WT. Taken together, our results suggest that SlAGO4A gene plays a negative role under salt and drought stress in tomato probably through the modulation of DNA methylation as well as the classical RNAi pathway. Hence, it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.     

Engineering plants for aphid resistance: current status and future perspectives

Key message

The current status of development of transgenic plants for improved aphid resistance, and the pros and cons of different strategies are reviewed and future perspectives are proposed.

Abstract

Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic plants engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. In this review, the distribution of major aphid species and their damages on crop plants, the so far isolated aphid-resistance genes and their applications in developments of transgenic plants for improved aphid resistance, and the pros and cons of these strategies are reviewed and future perspectives are proposed. Although the transgenic plants developed through expressing aphid-resistant genes, manipulating plant secondary metabolism and plant-mediated RNAi strategy have been demonstrated to confer improved aphid resistance to some degree. So far, no aphid-resistant transgenic crop plants have ever been commercialized. This commentary is intended to be a helpful insight into the generation and future commercialization of aphid-resistant transgenic crops in a global context.     

Role of insulin receptor and insulin signaling on αPS2CβPS integrins’ lateral diffusion

Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CβPS integrins’ lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24 % increase in the mobile integrin population, (2) 14 % of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45 % increase in the diameter of the confined zone, and (4) there was a 29 % increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.     

Growth and infectivity of callus cultures of tomato plants infected with a mycoplasma disease — Potato witches' broom

Callus tissues were derived from the stem of healthy tomato plants (Lycopersicum esculentum Mill. ev. Pr?honické) and of plants infected with potato witches' broom—a disease caused by mycoplasma. Callus cultures were established on modified fully synthetic media described byMorel (1948) and byMurashige andSkoog (1962). Callus cultures obtained from diseased plants were grown and subcultured on both media, growth in primary isolates from healthy plants took place on the Murashige and Skoog medium only. Growth of callus tissue derived from diseased plants was more vigorous even after several subcultivations in comparison with callus tissues isolated from healthy plants. Variations in the morphology in these callus cultures were not noted. Callus cells of diseased plants varied in size; they were about 50% larger than those from healthy ones. Implantation of primary and subcultivated callus tissues into tomato stems of healthy plants did not show any symptoms of infection on test plants.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.