Crispr/Cas9 Mediated Inactivation of Argonaute 2 Reveals its Differential Involvement in Antiviral Responses

Ludman, Márta; Burgyán, József; Fátyol, Károly

doi:10.1038/s41598-017-01050-6

Cited by 64 publications

(68 citation statements)

References 60 publications

(73 reference statements)

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“…Among the 10 AGO proteins encoded in the Arabidopsis thaliana genome, AGO2 has been identified most frequently as having antiviral function. Genetic ablation of AGO2 allows for increased accumulation of multiple wild‐type and VSR‐defective viruses in Arabidopsis (Carbonell & Carrington, ) and this function is conserved in the AGO2 homolog of Nicotiana benthamiana (Scholthof et al , ; Odokonyero et al , ; Fátyol et al , ; Ludman et al , ). Furthermore, AGO2 may play a role in determining virus host range.…”

Section: Introductionmentioning

confidence: 99%

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

et al. 2020

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Summary RNA silencing functions as an anti‐viral defence in plants through the action of DICER‐like (DCL) and ARGONAUTE (AGO) proteins. Despite the importance of this mechanism, little is known about the functional consequences of variation in genes encoding RNA silencing components. The AGO2 protein has been shown to be important for defense against multiple viruses, and we investigated how naturally occurring differences in AGO2 between and within species affects its antiviral activities. We find that the AGO2 protein from Arabidopsis thaliana, but not Nicotiana benthamiana, effectively limits potato virus X (PVX). Consistent with this, we find that the A. thaliana AGO2 gene shows a high incidence of polymorphisms between accessions, with evidence of selective pressure. Using functional analyses, we identify polymorphisms that specifically affect AGO2 antiviral activity, without interfering with other AGO2‐associated functions such as anti‐bacterial resistance or DNA methylation. Our results suggest that viruses adapt to overcome RNA silencing in their hosts. Furthermore, they indicate that plant–virus interactions have influenced natural variation in RNA‐silencing components and that the latter may be a source of genetically encoded virus resistance.

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Section: Introductionmentioning

confidence: 99%

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

et al. 2020

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“…Among the ten AGO proteins encoded by Arabidopsis thaliana , AGO2 has been identified most frequently as having antiviral function and this, against multiple different viruses 1 . Indeed, AGO2 appears to be a major determinant of the inability of PVX to systemically infect Arabidopsis accession Col-0 8 and an antiviral role for AGO2 is conserved in Nicotiana benthamiana 9,10,11,12 . However, despite the presence of a functional AGO2, N. benthamiana is susceptible to many viruses, including PVX 13 .…”

Section: Introductionmentioning

confidence: 99%

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

Brosseau

Adurogbangba

Roussin-Léveillée

et al. 2019

Preprint

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24RNA silencing functions as an anti-viral defence in plants through the action of DICER-like 25 (DCL) and ARGONAUTE (AGO) proteins. However, there are few known examples of 26 functional variation in RNA silencing components. The AGO2 protein is important for antiviral 27 defense against multiple viruses and has been shown to be a major limiting factor to infection 28 by potato virus X (PVX) of Arabidopsis thaliana but not Nicotiana benthamiana. We show that 29 the AGO2 proteins from these two plants have differential activity against PVX, suggesting that 30 variation in AGO2 is important in plant-virus interactions. Consistent with this, we find that the 31 Arabidopsis thaliana AGO2 gene shows a high incidence of polymorphisms between 32 accessions, with evidence of selective pressure. AGO2 protein variants can be assigned to two 33 groups, in near equal frequency, based on an amino acid change and small deletions in the 34 protein N-terminus. Inoculation of a large number of Arabidopsis accessions shows strong 35 correlation between these alleles and resistance or susceptibility to PVX. These observations 36 were validated using genetic and transgenic complementation analysis, which showed that one 37 type of AGO2 variant is specifically affected in its antiviral activity, without interfering with other 38 AGO2-associated functions such as anti-bacterial resistance or DNA methylation. Our results 39 demonstrate a novel type of genetically-encoded virus resistance and suggest that plant-virus 40 interactions have influenced natural variation in RNA silencing components. 41 42 43 44Here, we show that AGO2 proteins from A. thaliana and N. benthamiana manifest 68 differing activities against PVX, suggesting that inter-specific differences in AGO2 may 69 contribute to differing outcomes of PVX infection in these species. Furthermore, we tested 70 whether intra-specific differences in AGO2 might affect plant-virus interactions by taking 71 advantage of the natural genetic variation of wild Arabidopsis accessions. We show the AGO2 72 gene presents a high level of polymorphism and shows evidence of having been subject to 73 selective pressure. Furthermore, unlike the commonly used Arabidopsis accession Col-0, 27 74 out of 63 accessions analysed were found to susceptible to PVX. Through genetic and 75 transgenic analysis, we show that this susceptibility is determined by two polymorphisms found 76 in the N-terminus of the AGO2 protein. Our results have uncovered a novel form of genetically-77 encoded virus resistance. Likewise, it suggests that natural variation in AGO2 may be important 78 for determining plant-virus interaction outcomes and that in turn these pressures may have 79 shaped the RNA silencing machinery in ways similar to other defense mechanisms.80

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“…RNA silencing contributes to the regulation of gene expression and the maintenance of genome integrity in eukaryotes. In plants, nematodes, and insects, gene silencing is additionally an inducible, adaptable, specific, potent, and essential defense system against virus infection (antiviral RNA silencing) [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. Both endogenous and antiviral RNA silencing initiate with the processing of double-stranded RNA by Dicer-like (DCL) proteins to form small RNAs of 21 to 24 nt that associate with Argonaute (AGO) proteins to guide slicing or translational repression of target RNAs [ 2 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…Both endogenous and antiviral RNA silencing initiate with the processing of double-stranded RNA by Dicer-like (DCL) proteins to form small RNAs of 21 to 24 nt that associate with Argonaute (AGO) proteins to guide slicing or translational repression of target RNAs [ 2 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Sources of viral RNA that might trigger silencing include replication intermediates and self-complementary sequences in viral RNA, yielding primary virus-derived small interfering RNAs (siRNAs) that are necessary but not sufficient to restrict plant virus infection [ 1 ]. Restriction of plant virus infection requires silencing amplification by cellular RNA-dependent RNA polymerases (RDR) to establish an antiviral state [ 3 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant

García-Ruíz

Peralta

Harte-Maxwell

2018

Viruses

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Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana. Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection.

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Crispr/Cas9 Mediated Inactivation of Argonaute 2 Reveals its Differential Involvement in Antiviral Responses

Cited by 64 publications

References 60 publications

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant

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