Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity
Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B Hel , as well as their fuse...
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| Veröffentlicht in: | Frontiers in microbiology Jg. 15; S. 1451285 |
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| Abstract | Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (
Vitis
spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B
Hel
, as well as their fused form (1AB
Hel
). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B
Hel
in their systemic RNA silencing suppression ability by co-infiltrating
Nicotiana benthamiana
16c line plants with a
GFP
silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity
in planta
, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB
Hel
abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate
NbAGO2
expression by 1AB
Hel
. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of
NbDCL2
,
NbDCL4,
, and
NbRDR6
expression by 1B
Hel
. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection
in planta
. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate
NbDCL2
and
NbRDR6
expression. Finally,
in silico
protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. |
|---|---|
| AbstractList | Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B Hel , as well as their fused form (1AB Hel ). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B Hel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta , and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB Hel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1AB Hel . This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2 , NbDCL4, , and NbRDR6 expression by 1B Hel . This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta . Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B Hel , as well as their fused form (1AB Hel ). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B Hel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta , and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB Hel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1AB Hel . This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2 , NbDCL4, , and NbRDR6 expression by 1B Hel . This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta . Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B , as well as their fused form (1AB ). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B in their systemic RNA silencing suppression ability by co-infiltrating 16c line plants with a silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity , and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate expression by 1AB . This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of , , and expression by 1B . This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection . Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate and expression. Finally, protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. |
| Author | Choi, Jiyeong Schmitt-Keichinger, Corinne Browning, Scottie Fuchs, Marc |
| AuthorAffiliation | 3 INRAE, SVQV UMR 1131, Université de Strasbourg , Colmar , France 2 CNRS, IBMP UPR 2357, Université de Strasbourg , Strasbourg , France 1 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science College of Agriculture and Life Sciences, Cornell University, Cornell AgriTech at the New York State Agricultural Experiment Station , Geneva, NY , United States |
| AuthorAffiliation_xml | – name: 1 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science College of Agriculture and Life Sciences, Cornell University, Cornell AgriTech at the New York State Agricultural Experiment Station , Geneva, NY , United States – name: 2 CNRS, IBMP UPR 2357, Université de Strasbourg , Strasbourg , France – name: 3 INRAE, SVQV UMR 1131, Université de Strasbourg , Colmar , France |
| Author_xml | – sequence: 1 givenname: Jiyeong surname: Choi fullname: Choi, Jiyeong – sequence: 2 givenname: Scottie surname: Browning fullname: Browning, Scottie – sequence: 3 givenname: Corinne surname: Schmitt-Keichinger fullname: Schmitt-Keichinger, Corinne – sequence: 4 givenname: Marc surname: Fuchs fullname: Fuchs, Marc |
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| Copyright | Copyright © 2024 Choi, Browning, Schmitt-Keichinger and Fuchs. Attribution Copyright © 2024 Choi, Browning, Schmitt-Keichinger and Fuchs. 2024 Choi, Browning, Schmitt-Keichinger and Fuchs |
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| Keywords | WG/GW nepovirus AlphaFold2 plant-virus interaction RNA silencing silencing suppressor virus infectivity |
| Language | English |
| License | Copyright © 2024 Choi, Browning, Schmitt-Keichinger and Fuchs. Attribution: http://creativecommons.org/licenses/by This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
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Vitis... Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( spp.),... Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis... Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine ( Vitis... |
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| Title | Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity |
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