Polycistronic Artificial microRNA-Mediated Resistance to Cucumber Green Mottle Mosaic Virus in Cucumber

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.

Repression of barley cathepsins, HvPap-19 and HvPap-1, differentially alters grain composition and delays germination

During barley germination, cysteine-proteases are essential in the mobilization of storage compounds providing peptides and amino acids to sustain embryo growth until photosynthesis is completely established. Knock-down barley plants, generated by artificial microRNA, for the cathepsins B- and F-like, HvPap-19 and HvPap-1 genes, respectively, showed less cysteine protease activities and consequently lower protein degradation. The functional redundancy between proteases triggered an enzymatic compensation associated with an increase in serine-protease activities in both knock-down grains, which was not sufficient to maintain germination rates and behaviour. Concomitantly, these transgenic lines showed alterations in the accumulation of protein and carbohydrates in the grain. While the total amount of protein increased in both transgenic lines, the starch content decreased in HvPap-1 knock-down lines and the sucrose concentration was reduced in silenced HvPap-19 grains. Consequently, phenotypes of HvPap-1 and HvPap-19 artificial microRNA lines showed a delay in the grain germination process. These data expand the potential of exploring the properties of barley proteases to be selectively modified and used for brewing or livestock feeding industry.

Artificial microRNA-Based RNA Interference and Specific Gene Silencing for Developing Insect Resistance in Solanum lycopersicum

RNA Interference (RNAi), which works against invading nucleic acids or modulates the expression of endogenous genes, is a natural eukaryotic regulating system, and it works by noncoding smaller RNA molecules. Plant-mediated gene silencing through RNAi can be used to develop plants with insect tolerance at transcriptional or post-transcriptional levels. In this study, we selected Myzus persicae’s acetylcholinesterase 1 gene (Ace 1) as a silencing target to develop transgenic Solanum lycopersicum L. plants’ resistance to aphids. An RNAi plasmid vector containing an artificial microRNA (amiRNA) sequence was engineered and successfully transformed into Jamila and Tomaland, two elite tomato cultivars. A northern blot analysis and PCR were carried out to check the efficacy of Agrobacterium-mediated transformation in T0 transgenic plants. The quantitative PCR data showed a substantial downregulation of the Ace 1 gene in aphids fed in clip cages on T1 transgenic plants. Furthermore, there was a substantial drop in aphid colonies that were fed on T1 transgenic plants of both the cultivars. These findings strongly suggest that transgenic plants that express amiRNA could be an important tool for engineering plants resistant to aphids and possibly for the prevention of viral disease in other plant-infested pests.

Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs in Solanum lycopersicum and Nicotiana benthamiana

Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach; they have the advantage of producing just a single functional small RNA which can be designed for high target specificity and low off-target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans-acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA- and tasiRNA-based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5’-most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3’ end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single-target assays such as RNA blots or real-time PCR, is also discussed.Significance statementSeveral strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to fully analyze gene silencing experiments, and also the need for continued methodological development of these methods.

PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 modulates flowering in a florigen-independent manner by regulating SVP

ABSTRACTPHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) regulates phosphatidylethanolamine biosynthesis and controls the phosphatidylethanolamine:phosphatidylcholine ratio in Arabidopsis thaliana. Previous studies have suggested that PECT1 regulates flowering time by modulating the interaction between phosphatidylcholine and FLOWERING LOCUS T (FT), a florigen, in the shoot apical meristem (SAM). Here, we show that knockdown of PECT1 by artificial microRNA in the SAM (pFD::amiR-PECT1) accelerated flowering under inductive and even non-inductive conditions, in which FT transcription is almost absent, and in ft-10 twin sister of ft-1 double mutants under both conditions. Transcriptome analyses suggested that PECT1 affects flowering by regulating SHORT VEGETATIVE PHASE (SVP) and GIBBERELLIN 20 OXIDASE 2 (GA20ox2). SVP misexpression in the SAM suppressed the early flowering of pFD::amiR-PECT1 plants. pFD::amiR-PECT1 plants showed increased gibberellin (GA) levels in the SAM, concomitant with the reduction of REPRESSOR OF GA1-3 levels. Consistent with this, GA treatment had little effect on flowering time of pFD::amiR-PECT1 plants and the GA antagonist paclobutrazol strongly affected flowering in these plants. Together, these results suggest that PECT1 also regulates flowering time through a florigen-independent pathway, modulating SVP expression and thus regulating GA production.