Single-Dose Treatment With Vesicular Stomatitis Virus-Based Ebola Virus Vaccine Expressing Ebola Virus-Specific Artificial Micro-RNA Does Not Protect Mice From Lethal Disease.

Although significant progress has been made in the development of therapeutics against Ebola virus (EBOV), we sought to expand upon existing strategies and combine an RNA interference-based intervention with the approved vesicular stomatitis virus-based Ebola virus (VSV-EBOV) vaccine to conjointly treat and vaccinate patients during an outbreak. We constructed VSV-EBOV vectors expressing artificial micro-RNAs (amiRNAs) targeting sequences of EBOV proteins. In vitro experiments demonstrated a robust decrease in EBOV replication using a minigenome system and infectious virus. For in vivo evaluation, mouse-adapted EBOV-infected CD-1 mice were treated 24 hours after infection with a single dose of the VSV-EBOV amiRNA constructs. We observed no difference in disease progression or survival compared to the control-treated mice. In summary, while amiRNAs decrease viral replication in vitro, the effect is not sufficient to protect mice from lethal disease, and this therapeutic approach requires further optimization.

The lifetime of the oxygen-evolving complex subunit PSBO depends on light intensity and carbon availability in Chlamydomonas.

PSBO is essential for the assembly of the oxygen-evolving complex in plants and green algae. Despite its importance, we lack essential information on its lifetime and how it depends on the environmental conditions. We have generated nitrate-inducible PSBO amiRNA lines in the green alga Chlamydomonas reinhardtii. Transgenic strains grew normally under non-inducing conditions, and their photosynthetic performance was comparable to the control strain. Upon induction of the PSBO amiRNA constructs, cell division halted. In acetate-containing medium, cellular PSBO protein levels decreased by 60% within 24 h in the dark, by 75% in moderate light, and in high light, the protein completely degraded. Consequently, the photosynthetic apparatus became strongly damaged, probably due to 'donor-side-induced photoinhibition', and cellular ultrastructure was also severely affected. However, in the absence of acetate during induction, PSBO was remarkably stable at all light intensities and less substantial changes occurred in photosynthesis. Our results demonstrate that the lifetime of PSBO strongly depends on the light intensity and carbon availability, and thus, on the metabolic status of the cells. We also confirm that PSBO is required for photosystem II stability in C. reinhardtii and demonstrate that its specific loss also entails substantial changes in cell morphology and cell cycle.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.

Vector-delivered artificial miRNA effectively inhibits Porcine epidemic diarrhea virus replication.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV. METHODS: In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells. RESULTS: It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID50 results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID50 results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods. CONCLUSIONS: In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection.

An insight into microRNA biogenesis and its regulatory role in plant secondary metabolism.

KEY MESSAGE: The present review highlights the regulatory roles of microRNAs in plant secondary metabolism and focuses on different bioengineering strategies to modulate secondary metabolite content in plants. MicroRNAs (miRNAs) are the class of small endogenous, essential, non-coding RNAs that riboregulate the gene expression involved in various biological processes in most eukaryotes. MiRNAs has emerged as important regulators in plants that function by silencing target genes through cleavage or translational inhibition. These miRNAs plays an important role in a wide range of plant biological and metabolic processes, including plant development and various environmental response controls. Several important plant secondary metabolites like alkaloids, terpenoids, and phenolics are well studied for their function in plant defense against different types of pests and herbivores. Due to the presence of a wide range of biological and pharmaceutical properties of plant secondary metabolites, it is important to study the regulation of their biosynthetic pathways. The contribution of miRNAs in regulating plant secondary metabolism is not well explored. Recent advancements in molecular techniques have improved our knowledge in understanding the molecular function of genes, proteins, enzymes, and small RNAs involved in different steps of secondary metabolic pathways. In the present review, we have discussed the recent progress made on miRNA biogenesis, its regulation, and highlighted the current research developed in the field of identification, analysis, and characterizations of various miRNAs that regulate plant secondary metabolism. We have also discussed how different bioengineering strategies such as artificial miRNA (amiRNA), endogenous target mimicry, and CRISPR/Cas9 could be utilized to enhance the secondary metabolite production in plants.

Calumenin knockdown, by intronic artificial microRNA, to improve expression efficiency of the recombinant human coagulation factor IX.

OBJECTIVES: To improve the expression efficiency of recombinant hFIX, by enhancing its γ-carboxylation, which is inhibited by Calumenin (CALU), we used intronic artificial microRNAs (amiRNAs) for the CALU downregulation. METHODS: Two human CALU (hCALU)-specific amiRNAs were designed, validated and inserted within a truncated form of the hFIX intron 1, in either 3'- or 5'-untranslated regions of the hFIX cDNA, in an expression vector. After transfections of a human cell line with the recombinant constructs, processing of the miRNAs confirmed by RT-PCR, using stem-loop primers. The hFIX and hCALU expression assessments were done based on RT-PCR results. The Gamma(γ)-carboxylation of the expressed hFIX was examined by a barium citrate precipitation method, followed by Enzyme-Linked Immunosorbent Assay. RESULTS: Efficient CALU down regulations, with more than 30-fold decrease, occurred in the cells carrying either of the two examined the 3'-located amiRNAs. The CALU downregulation in the same cells doubled the FIX γ-carboxylation, although the transcription of the FIX decreased significantly. On the other hand, while the expression of the amiRNAs from the 5'-located intron had no decreasing effect on the expression level of CALU, the level of hFIX transcription in these cells increased almost twofold compared to the construct without amiRNA. CONCLUSION: The CALU downregulation, consistent with efficient hFIX γ-carboxylation, occurred in the cells carrying either of the two amiRNAs containing constructs, although it was affected by the locations of the amiRNA carrying introns, suggesting a possible need to optimize the conditions for the amiRNAs expression.

Characterization of Annexin gene family and functional analysis of RsANN1a involved in heat tolerance in radish (Raphanus sativus L.).

Plant annexins are a kind of conserved Ca2+-dependent phospholipid-binding proteins which are involved in plant growth, development and stress tolerance. Radish is an economically important annual or biennial root vegetable crop worldwide. However, the genome-wide characterization of annexin (RsANN) gene family remain largely unexplored in radish. In this study, a comprehensive identification of annexin gene family was performed at the whole genome level in radish. In total, ten RsANN genes were identified, and these putative RsANN proteins shared typical characteristics of the annexin family proteins. Phylogenetic analysis showed that the RsANNs together with annexin from Arabidopsis and rice were clustered into five groups with shared similar motif patterns. Chromosomal localization showed that these ten RsANN genes were distributed on six chromosomes (R3-R8) of radish. Several cis-elements involved in abiotic stress response were identified in the promoter regions of RsANN genes. Expression profile analysis indicated that the RsANN genes exhibited tissue-specific patterns at different growth stages and tissues. The Real-time quantitative PCR (RT-qPCR) revealed that the expression of most RsANN genes was induced under various abiotic stresses including heat, drought, salinity, oxidization and ABA stress. In addition, stress assays showed that overexpression of RsANN1a improved plant's growth and heat tolerance, while artificial microRNAs (amiRNA)-mediated knockdown of RsANN1a caused dramatically decreased survival ratio of Arabidopsis plants. These findings not only demonstrate that RsANN1a might play a critical role in the heat stress response of radish, but also facilitate clarifying the molecular mechanism of RsANN genes in regulating the biological process governing plant growth and development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01056-5.

Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance.

RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.

Research Tools for the Functional Genomics of Plant miRNAs During Zygotic and Somatic Embryogenesis.

During early plant embryogenesis, some of the most fundamental decisions on fate and identity are taken making it a fascinating process to study. It is no surprise that higher plant embryogenesis was intensively analysed during the last century, while somatic embryogenesis is probably the most studied regeneration model. Encoded by the MIRNA, short, single-stranded, non-coding miRNAs, are commonly present in all Eukaryotic genomes and are involved in the regulation of the gene expression during the essential developmental processes such as plant morphogenesis, hormone signaling, and developmental phase transition. During the last few years dedicated to miRNAs, analytical methods and tools have been developed, which have afforded new opportunities in functional analyses of plant miRNAs, including (i) databases for in silico analysis; (ii) miRNAs detection and expression approaches; (iii) reporter and sensor lines for a spatio-temporal analysis of the miRNA-target interactions; (iv) in situ hybridisation protocols; (v) artificial miRNAs; (vi) MIM and STTM lines to inhibit miRNA activity, and (vii) the target genes resistant to miRNA. Here, we attempted to summarise the toolbox for functional analysis of miRNAs during plant embryogenesis. In addition to characterising the described tools/methods, examples of the applications have been presented.

An artificial miRNA as a new tool to silence and explore gene functions in apple.

Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar 'Gala'. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

Genome-wide identification of the pectate lyase-like (PLL) gene family and functional analysis of two PLL genes in rice.

Pectate lyase catalyses the eliminative cleavage of de-esterified pectin, which is a major component of primary cell walls in many higher plants. Pectate lyase-like (PLL) genes have been identified in various plant species and are involved in a broad range of physiological processes associated with pectin degradation. Previous studies have functionally identified two PLL genes in rice (Oryza sativa. L). However, the knowledge concerning genome-wide analysis of this family remains limited, and functions of the other PLL genes have not been thoroughly elucidated to date. In this study, we identified 12 PLL genes based on a genome-wide investigation in rice. A complete overview of this gene family is presented, including chromosomal locations, exon-intron structure, cis-acting elements and conserved motifs. PLL protein sequences from multiple plant species were compared and divided into five groups based on phylogenetic analysis. Quantitative RT-PCR analysis revealed that only a portion of OsPLL genes (4 of 12) exhibits detectable expression levels. Notably, OsPLL1, OsPLL3, OsPLL4 and OsPLL12 exhibit strong and preferential expression in panicles suggesting that the potential roles of these genes are crucial during rice panicle development. Moreover, knockdown of OsPLL3 and OsPLL4 by artificial microRNA (amiRNA) disrupted normal pollen development and resulted in partial male sterility. These results could provide valuable information for characterising the functions and dissecting the molecular mechanisms of the OsPLL genes.

Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii.

Photosynthetic microbial hosts such as cyanobacteria and eukaryotic microalgae have recently emerged as alternative engineering platforms for the sustainable light-driven bio-production of terpenoids. Many desirable compounds with numerous applications can be produced in microorganisms by heterologous expression of terpene synthases. However, success of green microbial systems has been hampered by issues such as insufficient enzyme expression titers and low flux to desired terpenoid products from carbon fixed during photosynthesis. This work demonstrates how the green microalga Chlamydomonas reinhardtii can be engineered to produce the sesquiterpene biodiesel precursor (E)-α-bisabolene. Through strategic genetic engineering, substantial enhancements of productivity were achieved by coordinated tuning of the isoprenoid metabolism, combining serial enzyme loading for terpene synthase overexpression and amiRNA-based repression of competing pathways. Up to 10.3 ± 0.7mg bisabolene·g-1 cell dry weight could be produced in five days, which represents more than a 15-fold increase over single synthase expression strains. Investigation of strain performance in scale-up cultivations determined overall bisabolene productivity benefits from light:dark cycles. Mixotrophic cultivation can yield up to 11.0 ± 0.5mg bisabolene per liter in seven days in these conditions, and phototrophic production of 3.9 ± 0.2mg per liter was feasible. These achievements represent an important milestone in the engineering of C. reinhardtii towards the goal of designing sustainable, light-driven, green-cell algal bio-factories.

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi.

Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme.

Artificial miRNA-mediated down-regulation of two monolignoid biosynthetic genes (C3H and F5H) cause reduction in lignin content in jute.

Artificial microRNAs (amiRNA) provide a new feature in the gene silencing era. Concomitantly, reducing the amount of lignin in fiber-yielding plants such as jute holds significant commercial and environmental potential, since this amount is inversely proportional to the quality of the fiber. The present study aimed at reducing the lignin content in jute, by introducing amiRNA based vectors for down-regulation of two monolignoid biosynthetic genes of jute, coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H). The transgenic lines of F5H-amiRNA and C3H-amiRNA showed a reduced level of gene expression, which resulted in about 25% reduction in acid insoluble lignin content for whole stem and 12-15% reduction in fiber lignin as compared to the non-transgenic plants. The results indicate successful F5H-amiRNA and C3H-amiRNA transgenesis for lignin reduction in jute. This is likely to have far-reaching commercial implications and economic acceleration for jute producing countries.

Suppression of toxic transgene expression by optimized artificial miRNAs increases AAV vector yields in HEK-293 cells.

Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene's function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.

Efficient artificial microRNA-mediated resistance against zucchini yellow mosaic virus in zucchini via agroinfiltration.

Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

Trans-kingdom expression of an insect endogenous microRNA in rice enhances resistance to striped stem borer Chilo suppressalis.

BACKGROUND: The striped stem borer (SSB), Chilo suppressalis Walker, is a major pest of rice worldwide. Breeding of transgenic rice expressing Bacillus thuringiensis (Bt) toxins is a powerful strategy to control SSB. However, pests may evolve certain resistance to Bt toxins in transgenic plants. Hence, new controlling strategies must be continuously developed. RESULTS: We successfully generated SSB-resistant rice (csu-53) expressing the artificial microRNA (amiRNA) of SSB endogenous miRNA (csu-novel-miR53) through the RNAi-based technology. Feeding assays demonstrated that csu-53 rice inhibited larval growth, delayed pupation time, and reduced pupal weight and eclosion rate of SSB larva. In a 10-day feeding experiment, the miRNA mimic of csu-novel-miR53 also suppressed larval growth and more importantly increased larval mortality. Transcriptome analysis identified 28 differentially expressed unigenes (DEGs) in the midgut between SSB larvae fed on csu-53 rice and the wild type. One DEG (DN90065_c0_g12) validated by qRT-PCR had a predicted target site of csu-novel-miR53. In addition, in vitro double-stranded RNA synthesis and further feeding assay proved that DN90065_c0_g12 is most likely the target of csu-novel-miR53. CONCLUSION: amiRNA-mediated strategy can be applied to the development of insect-resistant crops, and the novel amiRNA csu-novel-miR53 of SSB has important application potential in developing SSB resistant rice. © 2021 Society of Chemical Industry.

Advances in RNA-Silencing-Related Resistance against Viruses in Potato.

Potato is a major food crop that has the potential to feed the increasing global population. Potato is the fourth most important crop and a staple food for many people worldwide. The traditional breeding of potato poses many challenges because of its autotetraploid nature and its tendency toward inbreeding depression. Moreover, potato crops suffer considerable production losses because of infections caused by plant viruses. In this context, RNA silencing technology has been successfully applied in model and crop species. In this review, we describe the RNA interference (RNAi) mechanisms, including small-interfering RNA, microRNA, and artificial microRNA, which may be used to engineer resistance against potato viruses. We also explore the latest advances in the development of antiviral strategies to enhance resistance against potato virus X, potato virus Y, potato virus A, potato leafroll virus, and potato spindle tuber viroid. Furthermore, the challenges in RNAi that need to be overcome are described in this review. Altogether, this report would be insightful for the researchers attempting to understand the RNAi-mediated resistance against viruses in potato.

Targeting of genomic and negative-sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations.

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus-specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive-sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative-sense RNA strand is cleaved by the amiRNA-guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.