Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence

Dunker, Florian; Trutzenberg, Adriana; Rothenpieler, Jan S; Kuhn, Sarah; Pröls, Reinhard; Schreiber, Tom; Tissier, Alain; Hückelhoven, Ralph; Weiberg, Arne

doi:10.1101/689190

Cited by 4 publications

(10 citation statements)

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“…The present knowledge on plant EVs is consistent with the idea of an EV-mediated effector shuttle in ckRNAi [1,22,[48][49][50][51][52][53][54][55]. Here, we tested the possibility that HIGS also involves vesicle transport of dsRNA-derived sRNAs.…”

Section: Exosome-like Vesicles From Leaf Extracts Of Cyp3rna-expressisupporting

confidence: 70%

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: Exosome-like Vesicles From Leaf Extracts Of Cyp3rna-expressisupporting

confidence: 70%

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

Schlemmer

Weipert

Barth

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The Ha co-IP sequencing runs were treated similarly as described by Dunker et al (2020). After downloading the already trimmed runs with fastq-dump the runs were transcribed to fasta with fastq_to_fasta and collapsed using the fastx_collapser from the fastx toolkit v0.0.14 (Gordon & Hannon 2010).…”

Section: Filtering Of Srnasmentioning

confidence: 99%

“…In recent years many more sRNA effectors have been discovered in plant pathogenic fungi (Wang et al 2017b;Dubey et al 2019;Werner et al 2021), oomycetes (Dunker et al 2020), beneficial fungal endophytes (Šečić et al 2021), and bacteria (Ren et al 2019). Their function as effectors is often experimentally demonstrated by knock-out (KO) of Dicer-like (DCLs) genes in the microbe and/or Argonaute (AGO) genes in the host, as loss-of-function mutations correspondingly impair either the microbe's ability to produce sRNAs or the host's ability to recognize silencing signals (Weiberg et al 2013;Cai et al 2021), while arguably DCLs and AGOs also play important roles in virulence and immunity both in plants (Fang et al 2016) and fungi (Nicolás et al 2013, Gaffar et al 2019, independent of ckRNAi.…”

Section: Introductionmentioning

confidence: 99%

“…Further experimental evidence for sRNA effector activity is provided by detecting corresponding degraded target host mRNAs fragments using degradome sequencing, also referred to as parallel analysis of RNA ends (PARE, German et al 2008), and/or through recording their effect on pathogenicity upon artificial overexpression in the host (Weiberg et al 2013). An elegant strategy to detect ckRNAi is the introduction of short tandem target mimics (STTM) that provides artificial RNA target sequences such that a cognate sRNA rather forms a complex with the target mimic, thus preventing silencing activity on its natural target (Yan et al 2012;Zhang et al 2016;Dunker et al 2020). Dunker et al (2020) examined plant AtAGO1linked sRNAs of the oomycete pathogen Hyaloperonospora arabidopsidis (Ha) after infection of Arabidopsis.…”

Section: Introductionmentioning

confidence: 99%

“…An elegant strategy to detect ckRNAi is the introduction of short tandem target mimics (STTM) that provides artificial RNA target sequences such that a cognate sRNA rather forms a complex with the target mimic, thus preventing silencing activity on its natural target (Yan et al 2012;Zhang et al 2016;Dunker et al 2020). Dunker et al (2020) examined plant AtAGO1linked sRNAs of the oomycete pathogen Hyaloperonospora arabidopsidis (Ha) after infection of Arabidopsis. Three of these sRNAs were confirmed as enhancing the pathogenicity of the oomycete by employing the STTM technique (Dunker et al 2020).…”

Section: Introductionmentioning

confidence: 99%

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Analysis of codon usage and allele frequencies reveal the double-edged nature of cross-kingdom RNAi

Werner

Kopp‐Schneider

Kogel

2022

Preprint

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BackgroundIn recent years, a new class of small 21- to 24-nt-(s)RNAs has been discovered from microbial pathogens that interfere with their host’s gene expression during infection, reducing the host’s defence in a process called cross-kingdom RNA interference (ckRNAi). According to this model, microbial sRNAs should exert selection pressure on plants so that gene sequences that reduce complementarity to sRNAs are preferred. In this paper, we test this consequence of the ckRNA model by analyzing changes to target sequences considering codon usage and allele frequencies in the model system Arabidopsis thaliana (At) – Hyaloperonospora arabidopsidis (Ha) and Hordeum vulgare (Hv) – Fusarium graminearum (Fg). In both pathosystems, some selected sRNA and their corresponding target have been described and experimentally validated, while the lengthy methodology prevents the analysis of all discovered sRNAs. To expand the understanding of ckRNAi, we apply a new in silico approach that integrates the majority of sRNAs.ResultsWe calculated the probability (PCHS) that synonymous host plant codons in a predicted sRNA target region would show the same or stronger complementarity as actually observed and compared this probability to sets of virtual analogous sRNAs. For the sets of Ha and Fg sRNAs, there was a significant difference in codon usage in their plant gene target regions (for Ha: PCHS 24.9% lower than in the virtual sets; for Fg: PCHS 19.3% lower than in the virtual sets), but unexpectedly for both sets of microbial sRNA we found a tendency towards codons with an unexpectedly high complementarity. To distinguish between complementarity caused by balancing sRNA-gene coevolution and directional selection we estimated Wright’s F-statistic (FST), a measurement of population structure, in which positive deviations from the background indicate directional and negative deviations balancing selection at the respective loci. We found a negative correlation between PCHS and FST (p=0.03) in the At-Ha system indicating deviations from codon usage favoring complementarity are generally directionally selected.ConclusionThe directional selection of complementary codons in host plants suggests an evolutionary pressure to facilitate silencing by exogenous microbial sRNAs, which is not consistent with the anticipated biological role of pathogen sRNAs as exclusively effectors in cross-kingdom RNAi. To resolve this conflict, we propose an extended model in which microbial sRNAs are perceived by plants via RNA interference and, via coevolution, primarily help to fine-tune plant gene expression.

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Herbivore insect small RNA effector suppress plant defense by cross-kingdom gene silencing

Han,

Ji,

Zhang

et al. 2023

Preprint

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Herbivore insects deploy salivary effectors to manipulate the defense of their host plants, however, whether insect small RNAs (sRNAs) act as effectors to regulate plant-insect interaction is currently unclear. Here, we report that a microRNA (miR29-b) from the saliva of phloem-feeding insects can transfer into the host plant phloem and fine-tune the host defense. The salivary gland’s abundant miR29-b was produced by insect Dicer 1 and insect salivary exosome is involved in its transferring and releasing into the host plant. Insect miR29-b effector hijacks plant Argonaute 1 to silence host defense geneBcl-2-associated athanogene 4(BAG4). Silencing ofBAG4suppressed the expression of phenylalanine ammonia-lyase and the accumulation of salicylic acid (SA), therefore negatively regulating host defense against herbivore insects. miR29-b is highly conserved in Hemiptera, Coleoptera, Hymenoptera, Orthoptera, and Blattaria insects and also targets theBAG4gene. Notably,BAG4orthologs exist in a wide range of plant species and may as the target of insect miR29-b. Our work provides new insight into the intriguing defense and counter-defense between herbivores and plants.TeaserPhloem-feeding insects produce and transfer small RNA into the host plants to fine-tune plant basal defense by cross-kingdom gene silencing.

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Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence

Abstract: 1 3

Cited by 4 publications

References 55 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

Analysis of codon usage and allele frequencies reveal the double-edged nature of cross-kingdom RNAi

Herbivore insect small RNA effector suppress plant defense by cross-kingdom gene silencing

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