Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing

Castro, Álvaro; Quiroz, Daniela; Sanchez, Evelyn Aparecida Mecenero; Miccono, María; Aguirre, Carlos; Ramírez, Alejandra Salas; Montes, Christian; Prieto, Humberto

doi:10.1016/j.jbiotec.2016.06.028

Cited by 11 publications

(3 citation statements)

References 30 publications

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“…End-point looped RT-PCR was used to detect the target small RNA species derived from NGS analyses. Stem-loop primers were designed for both miRNAs and siRNA according to Castro et al [54], using the “Stem-loop primer designer” option from the “amiRNA designer” tool available in the web page www.fruit-tree.genomics.com, tab “Biotools”. Primer sequences are presented in Additional file 8: Table S2.…”

Section: Methodsmentioning

confidence: 99%

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production

et al. 2019

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BackgroundIn plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV).ResultsFor this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka.ConclusionsOur results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

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Section: Methodsmentioning

confidence: 99%

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production

et al. 2019

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“…Ideally, the majority of reads from the asRNA gene correspond to the correctly processed asRNA; for example, in the case of the 21‐nt amiRNAs from the ath‐MIR390a ‐based precursors in Carbonell et al (2014). In other cases, the asRNA gene produces reads corresponding to the desired asRNA but also additional sRNAs from other regions of the precursor that accumulate at high levels and have potential unintended gene targets; for example, in the case of the ath‐MIR319a precursor in petunia and the vv‐MIR319e precursor in N. benthamiana (Castro et al , 2016; Guo et al , 2014).…”

Section: Introductionmentioning

confidence: 99%

Expression and processing of polycistronic artificial microRNAs and trans‐acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana

2021

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Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach, as 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. All analyses used transiently expressed transgenes delivered by infiltration of leaves with Agrobacterium tumefacians. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5 0 -most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3 0 -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 polymerase chain reaction, is also discussed.

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“…In this regard, the availability of genome drafts in several F&N species allow for the advance toward improved and safer application of techniques such as RNAi and CRISPR/Cas editing. Interference of target mRNAs using artificial microRNAs, or editing of on-target and effect over off-target loci can be assessed by ex-ante analyses using dedicated designers which predict these activities in an efficient rate (Doench et al, 2014; Castro et al, 2016). For instance, computing of protospacer adjacent motifs (PAMs) for Cas 9 over a target genome allows for a first-dimension analysis of the putative cut sites for the nuclease upon gRNA leadership.…”

Section: Recent Developments In Genetic Engineering Of Fandn Cropsmentioning

confidence: 99%

Agrobacterium-Mediated Transformation of Tree Fruit Crops: Methods, Progress, and Challenges

2019

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Genetic engineering based on Agrobacterium -mediated transformation has been a desirable tool to manipulate single or multiple genes of existing genotypes of woody fruit crops, for which conventional breeding is a difficult and lengthy process due to heterozygosity, sexual incompatibility, juvenility, or a lack of natural sources. To date, successful transformation has been reported for many fruit crops. We review the major progress in genetic transformation of these fruit crops made in the past 5 years, emphasizing reproducible transformation protocols as well as the strategies that have been tested in fruit crops. While direct transformation of scion cultivars was mostly used for fruit quality improvement, biotic and abiotic tolerance, and functional gene analysis, transgrafting on genetically modified (GM) rootstocks showed a potential to produce non-GM fruit products. More recently, genome editing technology has demonstrated a potential for gene(s) manipulation of several fruit crops. However, substantial efforts are still needed to produce plants from gene-edited cells, for which tremendous challenge remains in the context of either cell’s recalcitrance to regeneration or inefficient gene-editing due to their polyploidy. We propose that effective transient transformation and efficient regeneration are the key for future utilization of genome editing technologies for improvement of fruit crops.

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Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing

Cited by 11 publications

References 30 publications

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production

Expression and processing of polycistronic artificial microRNAs and trans‐acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana

Agrobacterium-Mediated Transformation of Tree Fruit Crops: Methods, Progress, and Challenges

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