siRNA-Finder (si-Fi) Software for RNAi-Target Design and Off-Target Prediction

Lück, Stefanie; Kreszies, Tino; Strickert, Marc; Schweizer, Patrick; Kuhlmann, Markus; Douchkov, Dimitar

doi:10.3389/fpls.2019.01023

Cited by 101 publications

(82 citation statements)

References 58 publications

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“…To explore the potential function for the wheat homologs of AtMin7 in the F. graminearum - wheat interaction we tested the effect of silencing the three homoeologous wheat genes, TraesCS2A02G202900, TraesCS2B02G230000 and TraesCS2D02G212800 , using Barley stripe mosaic virus- mediated Virus induced gene silencing (VIGS) on the FHB disease development (Lee et al, 2012). A 209-nt fragment highly conserved between coding sequences of the three TaMin7 homoeologs was selected as a target for VIGS using si-Fi21 software (Lück et al, 2019). This target fragment was predicted to generate a high number of silencing-effective siRNAs ( n = 91), and a very low likelihood of off-target silencing.…”

Section: Resultsmentioning

confidence: 99%

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection

Wood

Panwar

Grimwade-Mann

et al. 2020

Preprint

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Plants have developed intricate defense mechanisms, referred to as innate immunity, to defend themselves against a wide range of pathogens. Plants often respond rapidly to pathogen attack by the synthesis and delivery of various antimicrobial compounds, proteins and small RNA in membrane vesicles to the primary infection sites. Much of the evidence regarding the importance of vesicular trafficking in plant-pathogen interactions comes from the studies involving model plants whereas this process is relatively understudied in crop plants. Here we assessed whether the vesicular trafficking system components previously implicated in immunity in Arabidopsis thaliana play a role in the interaction with Fusarium graminearum, a fungal pathogen notoriously famous for its ability to cause Fusarium head blight (FHB) disease in wheat. Among the analyzed vesicular trafficking mutants, two independent T-DNA insertion mutants in the AtMin7 gene displayed a markedly enhanced susceptibility to F. graminearum. Earlier studies identified this gene, encoding an ARF-GEF protein, as a target for the HopM1 effector of the bacterial pathogen Pseudomonas syringae pv. tomato, which destabilizes AtMIN7 leading to its degradation and weakening host defenses. To test whether this key vesicular trafficking component may also contribute to defense in crop plants, we identified the candidate TaMin7 genes in wheat and knocked-down their expression through Virus induced gene silencing. Wheat plants in which TaMIN7 were silenced displayed significantly more FHB disease. This suggests that disruption of MIN7 function in both model and crop plants compromises the trafficking of innate immunity signals or products resulting in hyper-susceptibility to various pathogens.One sentence summaryDisruption of an ARF-GEF protein encoding gene AtMin7 in Arabidopsis thaliana and silencing of the orthologous gene in wheat result in hyper susceptibility to the fungal pathogen Fusarium graminearum.

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Section: Resultsmentioning

confidence: 99%

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection

Wood

Panwar

Grimwade-Mann

et al. 2020

Preprint

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“…hordei β2-tub gene (Supplementary File 1). The β2-tub-RNAi sequence used in this study was analyzed for possible off-targets in both formae speciales and their host species wheat and barley using the si-Fi software, which predicted no off-targets in either of the analyzed B. graminis and host genomes (Lück et al, 2019).…”

Section: Establishing Higs Of Bg Tritici Genes In Wheatmentioning

confidence: 99%

“…We selected a 500-nt fragment of the SvrPm3 a1/f 1 mRNA, which includes a segment of the 5 UTR and both exons excluding the last two codons (Figure 2A). Off-target analysis using the si-Fi software (Lück et al, 2019) predicted four putative siRNAs that map to a single 30-nt offtarget locus in a putative pseudogene on the wheat chromosome 6A. However, these siRNAs are predicted to be inefficient in directing silencing (Supplementary Note 1 and Supplementary File 2).…”

Section: Generation Of Stable Svrpm3 A1/f 1 -Rnai Wheat Linesmentioning

confidence: 99%

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat

et al. 2020

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Cross-kingdom RNA interference (RNAi) is a biological process allowing plants to transfer small regulatory RNAs to invading pathogens to trigger the silencing of target virulence genes. Transient assays in cereal powdery mildews suggest that silencing of one or two effectors could lead to near loss of virulence, but evidence from stable RNAi lines is lacking. We established transient host-induced gene silencing (HIGS) in wheat, and demonstrate that targeting an essential housekeeping gene in the wheat powdery mildew pathogen (Blumeria graminis f. sp. tritici) results in significant reduction of virulence at an early stage of infection. We generated stable transgenic RNAi wheat lines encoding a HIGS construct simultaneously silencing three B.g. tritici effectors including SvrPm3 a1/f 1 , a virulence factor involved in the suppression of the Pm3 powdery mildew resistance gene. We show that all targeted effectors are effectively downregulated by HIGS, resulting in reduced fungal virulence on adult wheat plants. Our findings demonstrate that stable HIGS of effector genes can lead to quantitative gain of resistance without major pleiotropic effects in wheat.

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“…The technique of RNAi in the medical field is expected to have not only therapeutic effects for human induced by knock-down specific genes but also suffers from off-target effects. Previous study reported that algorithm or open-source desktop software was developed to design RNAi sequences to exert strong and selective suppression of target genes and predict off-target [45,46]. However, it is difficult to predict specific side effects that appear due to off-target effects in human.…”

Section: Expected Side Effect Caused By Gene Therapy and Limitation Omentioning

confidence: 99%

Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine Peritoneal Dissemination Model

Hazekawa¹,

Nishinakagawa²,

Kawakubo‐Yasukochi³

et al. 2020

Gynaecological Malignancies - Updates and Advances

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Ovarian cancer is known to be the most lethal gynecologic cancer. It has been reported that Glypican-3 (Gpc3) expression induces immune responses, promotes the progression in ovarian cancer. Then, we focused on this Gpc3 gene silencing, tried to prepare siRNA delivery system. In this chapter, we introduce one of the therapeutic proposals in terms of novel drug delivery system using siRNA as a targeting medicine. This chapter introduces our works about preparation of siRNA-PLGA hybrid micelles to deliver the siRNA into the ovarian cancer cells and to evaluate gene silencing effects in mice model. As a result, siRNA-PLGA hybrid micelles were shown to effectively inhibit Gpc3 expression in vitro. In addition, siRNA-PLGA hybrid micelles also decreased the number of tumor nodes in the mesentery in vivo. These results suggested that Gpc3 could be a target molecule for ovarian cancer treatment and siRNA-PLGA hybrid micelles could be an effective siRNA delivery tool even in vivo.

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siRNA-Finder (si-Fi) Software for RNAi-Target Design and Off-Target Prediction

Cited by 101 publications

References 58 publications

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat

Therapeutic Effect of Glypican-3 Gene Silencing Using siRNA for Ovarian Cancer in a Murine Peritoneal Dissemination Model

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