Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

BACKGROUND: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ∼ 0.5 µM) was engineered to express a variant AtEPSPS-T102 I, P106 A (TIPA Ki = 150 µM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. RESULTS: Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha-1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. CONCLUSIONS: A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Analysis of plant-derived miRNAs in animal small RNA datasets.

BACKGROUND: Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms. RESULTS: To address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs. CONCLUSIONS: Our analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.

RNA decoys: an emerging component of plant regulatory networks?

The role of non-coding RNAs (ncRNAs), both short and long ncRNAs, in the regulation of gene expression has become evident in recent years. Non-coding RNA-based regulation is achieved through a variety of mechanisms; some are relatively well-characterized, while others are much less understood. MicroRNAs (miRNAs), a class of endogenous small RNAs, function as master regulators of gene expression in eukaryotic organisms. A notable, recently discovered role for long ncRNAs is that of miRNA decoys, also referred to as target mimics or sponges, in which long ncRNAs carry a short stretch of sequence sharing homology to miRNA-binding sites in endogenous targets. As a consequence, miRNA decoys are able to sequester and inactivate miRNA function. Engineered miRNA decoys are also efficacious and useful tools for studying gene function. We recently demonstrated that the potential of miRNA decoys to inactivate miRNAs in the model plants Arabidopsis thaliana and Nicotiana benthamiana is dependent on the level of sequence complementarity to miRNAs of interest. The flexibility of the miRNA decoy approach in sequence-dependent miRNA inactivation, backbone choice, ability to simultaneously inactivate multiple miRNAs, and more importantly, to achieve a desirable level of miRNA inactivation, makes it a potentially useful tool for crop improvement. This research addendum reports the functional extension of miRNA decoys from model plants to crops. Furthermore, endogenous miRNA decoys, first described in plants, have been proposed to play a significant role in regulating the transcriptome in eukaryotes. Using computational analysis, we have identified numerous endogenous sequences with potential miRNA decoy activity for conserved miRNAs in several plant species. Our data suggest that endogenous miRNA decoys can be widespread in plants and may be a component of the global gene expression regulatory network in plants.