Active 5' splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes.

Arabidopsis, miR402 that is encoded within the first intron of a protein-coding gene At1g77230, is induced by heat stress. Its upregulation correlates with splicing inhibition and intronic proximal polyA site selection. It suggests that miR402 is not processed from an intron, but rather from a shorter transcript after selection of the proximal polyA site within this intron. Recently, introns and active 5' splice sites (5'ss') have been shown to stimulate the accumulation of miRNAs encoded within the first exons of intron-containing MIR genes. In contrast, we have observed the opposite effect of splicing inhibition on intronic miR402 production. Transient expression experiments performed in tobacco leaves revealed a significant accumulation of the intronic mature miR402 when the 5'ss of the miR402-hosting intron was inactivated. In contrast, when the miR402 stem-loop structure was moved into the first exon, mutation of the first-intron 5'ss resulted in a decrease in the miRNA level. Thus, the 5'ss controls the efficiency of miRNA biogenesis. We also show that the SERRATE protein (a key component of the plant microprocessor) colocalizes and interacts with several U1 snRNP auxiliary proteins. We postulate that SERRATE-spliceosome connections have a direct effect on miRNA maturation.

Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses.

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula × tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence.

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Posttranscriptional coordination of splicing and miRNA biogenesis in plants.

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play a crucial role in basic physiological and morphological processes and in response to various stresses in eukaryotic organisms. However, the miRNA biogenesis, which is based on the action of complex protein machinery, varies between plants and animals, with the differences largely concerning the location of the process, the protein composition of the microprocessor, the mechanism of miRNA action on mRNA target, and the miRNA gene (MIR) structure. Roughly half of known Arabidopsis MIRs contain introns, and 29 miRNAs are encoded within the introns of host genes. Selection of alternative transcription start sites, alternative splice sites (SSs), and polyadenylation sites has been identified within miRNA primary transcripts (pri-miRNAs), and such variety is essential for the production and fine-tuning of miRNA levels. For example, the posttranscriptional processing of intron-containing pri-miRNAs involves the action of additional RNA metabolism machineries, such as the spliceosome and polyadenylation machinery, and to a large extent is based on direct communication between SERRATE (one of the core components of the plant microprocessor) and U1 snRNP auxiliary proteins. Moreover, the position of the miRNA stem-loop structure relative to the closest active 5'SS is essential for the miRNA production efficiency. Indeed, it is highly probable that this pre-miRNA location affects recruitment of the microprocessor to pri-miRNAs and therefore influences miRNA maturation and target mRNA regulation. Such complicated crosstalk between several machineries is important for a proper miRNA-connected response to biotic and abiotic stresses, ensuring plant survival in a changing environment. WIREs RNA 2017, 8:e1403. doi: 10.1002/wrna.1403 For further resources related to this article, please visit the WIREs website.

Virus-Induced Gene Silencing for Gene Function Studies in Barley.

Virus-Induced Gene Silencing (VIGS) creates a natural antiviral defense in plants. However, it has been also a powerful tool for endogenous gene silencing in dicot and monocot plants by exploitation of recombinant viruses, harboring silencing inducing sequences. The Barley Stripe Mosaic Virus (BSMV) based VIGS system is an efficient and rapid RNAi approach that is routinely applied in functional genomics studies of cereals. We present here a protocol for BSMV VIGS application in barley based on mechanical inoculation of the plants with in vitro transcribed recombinant BSMV RNAs as the silencing triggers.

Arabidopsis thaliana microRNA162 level is posttranscriptionally regulated via splicing and polyadenylation site selection.

Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level.

Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley.

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge - IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 - PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression.

Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses.

Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species.

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat.

BACKGROUND: Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species. RESULTS: Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-ß-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component. CONCLUSIONS: Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley.

We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley.

Determinants of plant U12-dependent intron splicing efficiency.

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated.