RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 α-Demethylase

Koch, Aline; Stein, Elke; Kogel, Karl‐Heinz

doi:10.1007/s10658-018-1518-4

Cited by 28 publications

(26 citation statements)

References 31 publications

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“…Moreover, these plants induced high levels of FgCYP51A silencing and FgCYP51B cosilencing, while FgCYP51C expression was not silenced (Figure 3(e)). This observation is consistent with the recent finding that CYP51-dsRNA constructs that target one or two FgCYP51 genes induce co-silencing [47]. However, co-silencing of FgCYP51B can be explained by off-target hits of siRNA-derived from Hotspot-A fragment whereas no off-targets were detected for FgCYP51C (Figure 3(f)).…”

Section: Evs From Apoplastic Fluids Of Transgenic Arabidopsis Containsupporting

confidence: 92%

“…Moreover, using transgenic Arabidopsis expressing the dsRNA CYP3RNA we demonstrate that EVs carry transgene-derived siRNA cargo ( Figure 2). Previous work showed that the 791 nt long CYP3RNA, when expressed in Arabidopsis or barley, inhibits the infection Fg by targeting the transcripts of its three FgCYP51 genes via RNAi [12,47,71]. Together these findings support the view that HIGS includes processing of dsRNA in the plant, incorporation of the produced siRNA into intracellular vesicles, secretion of the vesicles into the apoplastic space, and a potential uptake of the vesicles or their cargo into the interacting fungus ( Figure 6).…”

Section: Discussionsupporting

confidence: 74%

“…Here we assess whether artificial, antifungal dsRNA originating either from an endogenous transgene or exogenous spray application are incorporated into plant EVs. We show that EVs from leaves of transgenic Arabidopsis plants expressing CYP3RNA, a 791 nts long noncoding dsRNA that concomitantly silences the two fungal cytochrome P450 sterol 14α-demethylase genes CYP51A and CYP51B and the virulence factor CYP51C and inhibits the growth of Fusarium graminearum (Fg) both in vitro and in planta [12,25,47], contain CYP3RNA-derived siRNAs. Moreover, mutants of secretory vesicle pathway showed a loss of HIGS-mediated resistance.…”

Section: Caenorhabditis Elegans Sid Proteins (Systemic Rna Interferenmentioning

confidence: 99%

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Host-induced gene silencing involves Arabidopsis ESCRT-III pathway for the transfer of dsRNA-derived siRNA

Schlemmer

Weipert

Barth

et al. 2020

Preprint

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Small (s)RNA molecules are crucial factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ckRNAi). Consistent with this recent knowledge, sRNAs and their doublestranded (ds)RNA precursors have been adopted to control diseases in crop plants through transgenic expression (host-induced gene silencing, HIGS) or exogenous application (sprayinduced gene silencing, SIGS). While these strategies proved to be effective, the mechanism of RNA transfer at the plant -pathogen interface is widely unresolved. Here we show that extracellular vesicles (EVs) purified from Arabidopsis (Arabidopsis thaliana) leaf extracts and apoplastic fluids contain transgene-derived sRNAs. EVs from plants expressing CYP3RNA, a 791 nt long dsRNA, which was originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. Notably, the EVs cargo retained the same CYP3RNA-derived siRNA profile as the respective leaf extracts, suggesting that there was no selective uptake of specific artificial sRNAs into EVs. In addition, mutants of the ESCRT-III complex were impaired in HIGS further indicating that endosomal vesicle trafficking supports transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells.Further supporting the relevance of EV-mediated transport of sRNA, we demonstrate that HIGS plants, expressing a 100 nt dsRNA-target-sequence identified via EV-sRNA-seq of CYP3RNA Arabidopsis, confers strong resistance to F. graminearum. Together, these findings support the view that EVs are key mediators in the transport of HIGS-related sRNAs to reduce the virulence of interacting fungal pathogens during host-pathogen interaction.(MVBs) fuse with the PM [33][34][35][36][37]. In mammals, exosomes mediate intercellular communication by shuttling proteins, lipids and RNAs, where RNA molecules remain functional after delivery and can elicit effects in the recipient cell [38][39][40].Based on this knowledge, we speculated that HIGS-and SIGS-related RNAs also are loaded into plant vesicles and transferred by EVs that cross the plant-fungus interface. It was reasoned already more than 50 years ago that a fusion of plant MVBs with the PM may result in the release of small vesicles into the extracellular space [41]. Consistent with this early observation, biogenesis of immune-related MVBs [29,30] and the release of their cargo via EVs also is inherent to the plant secretory pathway [27]. Transmission electron microscopy (TEM) revealed proliferation of MVBs next to plant cell wall papillae in response to infection with powdery mildew fungus and the release of para-mural vesicles. The authors proposed that released vesicles might be similar to exosomes in animal cell thus anticipating that exosomes exist in plants [29,30]. Immun...

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Section: Evs From Apoplastic Fluids Of Transgenic Arabidopsis Containsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 74%

Section: Caenorhabditis Elegans Sid Proteins (Systemic Rna Interferenmentioning

confidence: 99%

See 1 more Smart Citation

Host-induced gene silencing involves Arabidopsis ESCRT-III pathway for the transfer of dsRNA-derived siRNA

Schlemmer

Weipert

Barth

et al. 2020

Preprint

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“…In addition to the generation of RNA silencing signals in planta , plants can be protected from pathogens and pests by exogenously applied RNA biopesticides (known as spray-induced gene silencing, SIGS) (for review see: Mitter et al 2017; Cai et al 2018; Dubrovina and Kiselev 2019; Gaffar and Koch 2019; Dalakouras et al 2020). Over the last decade, our research (Koch et al, 2013; 2016; 2018; 2019; Höfle et al, 2019, Werner et al, 2019) and the work of others have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection, regardless of how target-specific inhibitory RNAs are applied (i.e. endogenously or exogenously).…”

Section: Introductionmentioning

confidence: 99%

Phloem-mediated spreading of SIGS-derived non-coding RNAs inHordeum vulgare

Biedenkopf

Will

Knauer³

et al. 2019

Preprint

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1 7 Highlight 1 8The use of the SIGS-technology as a biopesticide will require the systemic spreading of 1 9 dsRNA/siRNA signals. Our findings strongly support the notion of phloem-mediated long-2 0 distance movement of SIGS-associated RNAs. Abstract 2 2 Small (s)RNA molecules are crucial factors in the communication between hosts and their 2 3 3 1 distance movement of SIGS-associated dsRNA and/or siRNA. These findings are significant 3 2 contributions to our mechanistic understanding of RNA spray technology, as our previous 3 3 data indicate that SIGS requires the processing of dsRNAs by the fungal RNAi machinery. In 3 4 summary, our findings support the model that SIGS involves: (i) uptake of sprayed dsRNA by 3 5 the plant (via stomata); (ii) transfer of apoplastic dsRNAs into the symplast (DCL processing 3 6 into siRNAs); (iii) systemic translocation of siRNA or unprocessed dsRNA via the vascular 3 7 system (phloem/xylem); (iv) uptake of apoplastic dsRNA or symplastic dsRNA/siRNA 3 8 depending on the lifestyle/feeding behavior of the pathogen/pest.3 9

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“…Regardless of how target-specific inhibitory RNAs are applied (i.e. endogenously or exogenously), the use of HIGS and SIGS technologies to control Fusarium species have been shown to be a potential alternative to conventional pesticides (Koch et al 2013; Ghag et al 2014; Cheng et al 2015; Hu et al 2015; Chen et al 2016; Pareek and Rajam 2017; Bharti et al 2017; Baldwin et al 2018; Koch et al 2018; Koch et al 2019), supporting the notion that RNAi strategies may improve food safety by controlling the growth of phytopathogenic, mycotoxin-producing fungi (reviewed by Machado et al 2017; Majumdar et al 2017).…”

Section: Introductionmentioning

confidence: 99%

RNA-spray-mediated silencing ofFusarium graminearum AGOandDCLgenes improve barley disease resistance

Werner

Gaffar

Schuemann

et al. 2019

Preprint

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AbstractOver the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an in planta expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals in planta, plants can be protected from pathogens and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed versus manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

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RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 α-Demethylase

Abstract: RNA-based disease control has shown great potential for controlling pest and diseases in crop plants.

Cited by 28 publications

References 31 publications

Host-induced gene silencing involves Arabidopsis ESCRT-III pathway for the transfer of dsRNA-derived siRNA

Host-induced gene silencing involves Arabidopsis ESCRT-III pathway for the transfer of dsRNA-derived siRNA

Phloem-mediated spreading of SIGS-derived non-coding RNAs inHordeum vulgare

RNA-spray-mediated silencing ofFusarium graminearum AGOandDCLgenes improve barley disease resistance

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