Arch Virol DOI 10.1007/s00705-013-1928-8
BRIEF REPORT
Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae) Ying Zhai • Sudeep Bag • Neena Mitter Massimo Turina • Hanu R. Pappu
•
Received: 29 June 2013 / Accepted: 14 November 2013 Ó Springer-Verlag Wien 2013
Abstract Tospoviruses cause serious economic losses to a wide range of field and horticultural crops on a global scale. The NSs gene encoded by tospoviruses acts as a suppressor of host plant defense. We identified amino acid motifs that are conserved in all of the NSs proteins of tospoviruses for which the sequence is known. Using tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) at positions 181-183 and tyrosine and leucine (YL) at positions 412-413, and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, we show that substitutions (K182 to A, and L413 to A) in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the RNAi suppressor function of tospoviruses.
Thrips-transmitted tospoviruses (genus Tospovirus, family Bunyaviridae) constitute an economically important group
Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1928-8) contains supplementary material, which is available to authorized users. Y. Zhai S. Bag H. R. Pappu (&) Department of Plant Pathology, Washington State University, PO Box 646430, Pullman, WA 99164, USA e-mail: [email protected] N. Mitter QAAFI, University of Queensland, St Lucia, QLD, Australia M. Turina Istituto di Virologia Vegetale, CNR, Strada delle Cacce 75, 10135 Turin, Italy
of viruses affecting field and horticultural crops worldwide [18, 22, 27, 34]. New viruses, thrips species with vectoring capability, and new hosts continue to be reported, and more than 29 distinct tospoviruses have now been identified in different parts of the world. Tospoviruses share the following genomic features: three single-stranded (ss) RNAs: Large (L), Medium (M), and Small (S), with the L RNA in negative sense and the M and S RNAs arranged in an ambisense organization. The L RNA encodes the 331-kDa RNA-dependent RNA polymerase (RdRp). The M RNA encodes a non-structural movement protein, NSm, and the precursors of two structural glycoproteins, GN and GC, while the S RNA codes for the nucleocapsid protein N and another non-structural protein, NSs. The three genomic RNAs and the N protein are tightly linked to form ribonucleoproteins (RNPs), which are encapsulated by the glycoprotein envelope [1, 12, 20, 21, 29, 33]. Thrips play an important role in tospovirus dissemination, and a high degree of specificity exists that define thrips-tospovirus interactions [30, 35, 38]. Similar to most plant-infecting RNA viruses, the genome of tospoviruses codes for a gene that suppresses the host plant’s silencing mechanism. It has been shown that the NSs protein encoded by the NSs gene located on the S RNA of TSWV functions as a suppressor of posttranscriptional gene silencing, or RNA silencing, and also acts as an avirulence determinant [19, 32]. During viral infection, the plant cell recognizes the virus sequence, specifically the double-stranded RNA (dsRNA), which triggers the plant defense response or gene silencing [3, 7, 13, 36]. It has been widely accepted that most plant viruses carry a silencing suppressor gene [4]. Using several model systems, such as cucumber mosaic virus, potyviruses, and tomato bushy stunt virus, the mechanism of these virally encoded silencing suppressors has been elucidated [10, 25,
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Y. Zhai et al. Fig. 1 Alignment of the amino acid sequence of the NSs protein of tomato spotted wilt virus (TSWV) with those of other tospoviruses. (A) Alignment of amino acid sequences of NSs proteins between positions 150–200 and (B) between positions 400–450
37]. In many cases, it has been shown that these silencing suppressors interfere with RNA silencing through doublestranded RNA (dsRNA) binding, which can sequester siRNA duplexes, preventing the small RNA from being loaded into the Argonaut protein (AGO), which is the central catalytic component of RNA-induced silencing complex (RISC), thus interfering with the assembly of the RNA-induced silencing complex [9, 14]. In the case of TSWV, it has been shown that NSs can bind both long and short dsRNAs and interfere with Dicer cleavage [28]. In groundnut bud necrosis virus (GBNV), a tospovirus that is widely prevalent in the Indian subcontinent, the NSs protein has been shown to have NTPase activity and ATP-independent 50 DNA/RNA phosphatase activity [15]. Recently, it has been shown that there is a potential synergistic interaction between iris yellow spot virus and TSWV in doubly infected datura plants, resulting in increased accumulation of NSs of both viruses [2]. The importance of NSs in suppressing the plant defense mechanism underscores the need to gain a better understanding of the structure-function relationships of the NSs gene. However, little is known about the NSs regions that
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are critical in modulating suppressor activity. In the case of tospoviruses, structure-function studies have been hampered by the lack of a reverse genetics system. Most of the studies on identifying genetic determinants of symptom severity or vector transmission, or those assigning functions to various virally encoded genes have utilized naturally occurring or artificially generated virus variants with distinct biological properties. Here, we utilize a widely used ‘reversal-of-gene-silencing assay’ and site-directed mutations to investigate the importance of selected conserved regions of the NSs protein for their relative importance in maintaining suppressor activity. To identify the conserved regions of NSs that may be critical for its suppressor activity, the deduced amino acid sequence of the NSs protein of TSWV was aligned with those of fifteen other tospoviruses (Fig. 1A and B). These included calla lily chlorotic spot virus (CCSV), capsicum chlorosis virus (CaCV), chrysanthemum stem necrosis virus (CSNV), groundnut bud necrosis virus (GBNV), groundnut ringspot virus (GRSV)-African, groundnut ringspot virus (GRSV)-associated-Florida isolate, iris yellow spot virus (IYSV), impatiens necrotic spot virus
Mutational analysis of a tospovirus silencing suppressor
(INSV), melon yellow spot virus (MYSV), polygonum ringspot virus (PolRSV), tomato yellow ring virus (TYRV), tomato zonate spot virus (TZSV), watermelon bud necrosis virus (WBNV), watermelon silver mottle virus (WSMoV), and zucchini lethal chlorosis virus (ZLCV). Two motifs, GKV/T at positions 181-183 and YL at positions 412-413, were found to be conserved among all tospoviruses analyzed. Using a Quikchange Lightning sitedirected mutagenesis kit (Invitrogen, Santa Clara CA), sitedirected point mutations were made to change K 182 to A in the GKT motif (designated as NSs-1) and L 413 to A in the YL motif (designated as NSs-2). Primers for the sitedirected mutagenesis of the GKV motif (A473G_A474C and A473G_A474_antisense) and YL motif (t132g_t133c and t132g_t133c_antisense) were designed using the Agilent QuikChange primer design tool (Supplemental Table 1). The complete NSs gene was amplified by RT-PCR, cloned into cloning vector pART7 [11], and verified by DNA sequencing. The plasmid clone was digested with NotI to release the complete NSs expression cassette containing the CaMV 35S promoter. The NSs expression fragment was then cloned into binary vector pART27 [11] for agroinfiltration. We utilized a ‘‘reversal-of-silencing assay’’ (Agrobacterium co-infiltration assay) to evaluate and assess the silencing suppressor activity of wild-type and mutant constructs of TSWV NSs. The original GFP signal is generated by the GFP gene in transgenic N. benthamiana line 16c. Infiltration of pCAMBIA 1302 can abolish the GFP signal due to co-suppression. Coinfiltration of pCAMBIA1302 and wild-type NSs can suppress RNA silencing, thereby restoring the expression of GFP. Transgenic N. benthamiana line 16c expressing GFP [26], which exhibits green fluorescence under UV light, was used. Non-transgenic N. benthamiana leaves show red fluorescence under UV light due to chlorophyll. The plasmid pCAMBIA1302, harboring 35S:GFP, was used for silencing GFP expression in line 16c. Competent Agrobacterium tumefaciens strain GV3101 cells were electroporated separately with the recombinant plasmid containing the NSs wild type, NSs-1 and NSs-2 constructs. Bacterial cells were then plated on yeast extract-peptone (YEP) plates containing spectinomycin. N. benthamiana line 16c leaves were agroinfiltrated at the 5- to 6-leaf stage. A. tumefaciens was inoculated in 5 ml of YEP liquid medium containing spectinomycin and rifampicin and incubated for 20 h at 28 °C with vigorous shaking. A 2-ml culture was transferred to fresh 50 ml YEP liquid medium containing spectinomycin and rifampicin and grown for 20 h at 28 °C with vigorous shaking. A. tumefaciens cultures were centrifuged at 4000 rpm for 10 minutes, and the cells were resuspended in infiltration buffer (10 mM
MgCl2, 10 mM MES, pH 5.7, 2.25 mM acetosyringone). The volume of the culture was adjusted with the infiltration medium to a final optical density (OD) of 0.5 at 600 nm. Equal volumes of each A. tumefaciens culture were mixed. Before infiltration, the mixture was incubated in the dark without shaking for six hours [16]. After infiltration, the plants were maintained in a greenhouse (16 hours of light and eight hours of darkness). GFP fluorescence was detected using a 365-nm long-wave UV lamp. Leaves were photographed using a Panasonic Lumix digital camera. Leaves of GFP-expressing N. benthamiana line 16c agroinfiltrated with pCAMBIA 1302 alone do not exhibit GFP fluorescence due to co-suppression of GFP gene expression. However, co-infiltration of pCAMBIA1302 and the wild-type TSWV NSs construct restores GFP fluorescence due to the silencing suppressor activity of TSWV NSs. In the case of NSs-1 and NSs-2, in which selected amino acids were mutated, if these mutations affected the suppressor activity of NSs, restoration of GFP fluorescence would be expected to be diminished or completely abolished in leaves co-agroinfiltrated with either of these constructs along with pCAMBIA1302. The line 16c leaves co-agroinfiltrated with pCAMBIA1302 and the wild-type NSs construct showed expression of GFP under UV light, indicating silencing suppressor activity of TSWV NSs (Fig. 2A). Line 16c leaves infiltrated with pCAMBIA1302 alone as a negative control exhibited red fluorescence under UV light, as expected (Fig. 2B). The line 16c leaves co-agroinfiltrated with pCAMBIA1302 and TSWV NSs-1 or NSs-2 mutant did not show any GFP expression (Fig. 2C, D) indicating that the ability of NSs-1 to act as a suppressor was negatively affected as a consequence of these mutations. A double mutant consisting of NSs-1 and NSs-2 was made (referred to as NSs-3) and tested for its effect on NSs suppressor activity. When coagroinfiltrated with pCAMBIA1302, the NSs-3 mutant also failed to restore GFP expression, and only red fluorescence was observed (Fig. 2E). To confirm that the amino acid substitutions did not have any influence on the stability of the NSs protein, a direct antigen-coated-ELISA (DAC-ELISA) assay was performed on all the co-agroinfiltrated leaves using a monoclonal antibody that is specific for the TSWV NSs (Agdia, Elkhart IN). The N. benthamiana samples were ground in a 1:1 (w/v) dilution in carbonate buffer containing 2 % polyvinyl pyrrolidone (PVP, MW 40,000). The TSWV NSs antiserum was diluted (1:5000), and antimouse immunoglobulin alkaline phosphatase (universal conjugate, Sigma, USA) was diluted (1:20,000) in PBSTPO buffer. Absorbance values at 405 nm were recorded using a, BioTek ELX 800 microplate reader. TSWVinfected leaves were used as a positive control. The NSs protein could be detected by ELISA in leaves co-
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Fig. 2 Effect of point mutations on the silencing suppression activity of tomato spotted wilt virus (TSWV) NSs in Nicotiana benthamiana line 16c plants. A, leaf from N. benthamiana line 16c plants infiltrated with a mixture of pCAMBIA1302 and full-length TSWV NSs in a p7/ p27 construct. B, leaf from N. benthamiana line 16c plants infiltrated with pCAMBIA1302 and empty p7/p27 vector. C, leaf from N. benthamiana line 16c plants infiltrated with a mixture of pCAMBIA1302 and the NSs-1 construct (GKV motif mutant). D, leaf from N. benthamiana line 16c plants infiltrated with mixture of pCAMBIA1302 and the NSs-2 construct (YL motif mutant). E, leaf from N. benthamiana line 16c plants infiltrated with a mixture of
pCAMBIA1302 and double mutant (mutations in both the GKV and YL motifs). F, western blot analysis for NSs detection of three mutant constructs. Lane 1, N. benthamiana line 16c leaf infiltrated with empty p7/p27. Lane 2, TSWV-infected line 16c leaf as a positive control. Lane 3, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and the TSWV NSs full sequence (wild-type NSs) in a p7/ p27 construct. Lane 4, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and the NSs-1 construct. Lane 5, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and NSs-2 construct. Lane 6, line 16c leaf infiltrated with mixture of pCAMBIA1302 and NSs-3
agroinfiltrated with either the wild-type TSWV NSs or those with mutant constructs (data not shown). Western blotting was performed to verify the results of ELISA. The antisera used consisted of TSWV NSs-specific monoclonal antibody (Agdia Inc., Elkhart, IN) as the primary antibody (used at 1:5000 dilution) and anti-mouse immunoglobulin conjugated with alkaline phosphatase (Sigma; used at 1:20,000 dilution) as the secondary antibody. Protein bands were visualized using BCIP/NBT as substrates. The NSs protein could be detected in leaves agroinfiltrated with all three mutant constructs, confirming that the mutations did not affect the expression or stability of the protein (Fig. 2F). It has been shown that almost all plant viruses code for a suppressor of gene silencing [4]. Virally encoded suppressors act by interacting with dsRNA and interfering with the assembly of the RISC [14]. In the case of tospoviruses, it has
been shown that the NSs protein acts as the silencing suppressor [32] and is an avirulence determinant [19]. Among several possible mechanisms, it can interact with siRNA or dsRNA and prevent them from loading to the RISC [28]. A recent report suggested that the NSs of GBNV contains NTPase activity and ATP-independent 50 DNA/RNA phosphatase activity. The mutation of K189 to A in the GKT motif resulted in the complete loss of ATPase activity. However, the 50 phosphatase activity was not affected by the mutation [15]. Our results indicated that the conserved amino acids in TSWV NSs play a vital role in its suppressor function. Both N-terminal and C-terminal sequences are indispensable for the silencing suppressor function of the NSs protein. In our study, two constructs were made, each with either the N part (1-227 aa) or C part (245-467 aa) of the protein. Neither of the truncated proteins retained the suppressor activity (Fig. 3).
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Mutational analysis of a tospovirus silencing suppressor
Fig. 3 Effect of deletions in the NSs gene of tomato spotted wilt virus (TSWV) on its gene suppressor activity in Nicotiana benthamiana line 16c plants. A, leaf infiltrated with a mixture of pCAMBIA1302 and a full-length gene TSWV NSs sequence in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). B, leaf infiltrated with a mixture of pCAMBIA1302 and the truncated
gene containing the amino-terminal 227 aa of TSWV-NSs in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). C, leaf infiltrated with a mixture of pCAMBIA1302 and the carboxyl terminal aa 245-467 of TSWV-NSs in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). Arrows indicate the infiltrated area on the leaf
Amino acids that are crucial for protein function or binding capacity have been identified in tospovirus NSs proteins. For example, Cheng et al. [5] identified nine core amino acids from watermelon silver mottle virus (WSMoV) that interact with NSscon (23 aa; a common epitope in the gene silencing suppressor NSs proteins of the members of the WSMoV serogroup) monoclonal antibody. In our study, three conserved motifs were identified in the TSWV NSs protein: GKV (aa 181-183), KTL (aa 398-400), and YL (aa 412-413). In order to identify the functional motifs to which these conserved sites belong, the NSs protein sequence was scanned using protein function site prediction software (http://elm.eu.org/). The KTL region does not exist in any of the identified motifs and was not included in this investigation. The GKV region is within the motif recognized by the Src Homology 3 (SH3) domain, and the YL region is within the Src Homology 2 (SH2) domain binding motif (Supplemental Table 2). Past reports have revealed the importance of SH2 domain–SH2 binding motif and SH3 domain–SH3 binding motif interactions in virus replication and signal transduction pathways [6, 17, 23]. In one recent example from hepatitis C virus (HCV) [24], the Src family kinase c-Src was shown to interact directly with viral RdRp via its SH3 domain, and with nonstructural phosphoprotein NS5A via its SH2 domain. The SH2 and SH3 domains are essential for the interaction between c-Src and RNA-binding proteins [31] and the regulation of their RNA-binding ability [8]. Mutations in the GKV or YL regions caused the disruption of SH2- and SH3-binding motifs in NSs, which probably affected its RNA-binding ability and therefore suppressed its anti-RNAi function. Our findings provide the first experimental evidence for the role of selected regions that are critical for TSWV NSs suppressor activity. Information about the relative
importance of these conserved motifs could be useful in developing novel RNAi-based approaches for antiviral strategies. Acknowledgements This work was supported by the WSU Agricultural Research Center, PPNS No. 0627, Department of Plant Pathology, College of Agricultural, Human and Natural Resource Sciences, Agricultural Research Center, Project # WNPO 0545, Washington State University, Pullman, WA, 99164-6430, USA. HRP’s visit to Instituto Virologia Vegetale, CNR, Turin, Italy, was supported by a fellowship from the OECD under the Cooperative Research Program.
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BRIEF REPORT
Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae) Ying Zhai • Sudeep Bag • Neena Mitter Massimo Turina • Hanu R. Pappu
•
Received: 29 June 2013 / Accepted: 14 November 2013 Ó Springer-Verlag Wien 2013
Abstract Tospoviruses cause serious economic losses to a wide range of field and horticultural crops on a global scale. The NSs gene encoded by tospoviruses acts as a suppressor of host plant defense. We identified amino acid motifs that are conserved in all of the NSs proteins of tospoviruses for which the sequence is known. Using tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) at positions 181-183 and tyrosine and leucine (YL) at positions 412-413, and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, we show that substitutions (K182 to A, and L413 to A) in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the RNAi suppressor function of tospoviruses.
Thrips-transmitted tospoviruses (genus Tospovirus, family Bunyaviridae) constitute an economically important group
Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1928-8) contains supplementary material, which is available to authorized users. Y. Zhai S. Bag H. R. Pappu (&) Department of Plant Pathology, Washington State University, PO Box 646430, Pullman, WA 99164, USA e-mail: [email protected] N. Mitter QAAFI, University of Queensland, St Lucia, QLD, Australia M. Turina Istituto di Virologia Vegetale, CNR, Strada delle Cacce 75, 10135 Turin, Italy
of viruses affecting field and horticultural crops worldwide [18, 22, 27, 34]. New viruses, thrips species with vectoring capability, and new hosts continue to be reported, and more than 29 distinct tospoviruses have now been identified in different parts of the world. Tospoviruses share the following genomic features: three single-stranded (ss) RNAs: Large (L), Medium (M), and Small (S), with the L RNA in negative sense and the M and S RNAs arranged in an ambisense organization. The L RNA encodes the 331-kDa RNA-dependent RNA polymerase (RdRp). The M RNA encodes a non-structural movement protein, NSm, and the precursors of two structural glycoproteins, GN and GC, while the S RNA codes for the nucleocapsid protein N and another non-structural protein, NSs. The three genomic RNAs and the N protein are tightly linked to form ribonucleoproteins (RNPs), which are encapsulated by the glycoprotein envelope [1, 12, 20, 21, 29, 33]. Thrips play an important role in tospovirus dissemination, and a high degree of specificity exists that define thrips-tospovirus interactions [30, 35, 38]. Similar to most plant-infecting RNA viruses, the genome of tospoviruses codes for a gene that suppresses the host plant’s silencing mechanism. It has been shown that the NSs protein encoded by the NSs gene located on the S RNA of TSWV functions as a suppressor of posttranscriptional gene silencing, or RNA silencing, and also acts as an avirulence determinant [19, 32]. During viral infection, the plant cell recognizes the virus sequence, specifically the double-stranded RNA (dsRNA), which triggers the plant defense response or gene silencing [3, 7, 13, 36]. It has been widely accepted that most plant viruses carry a silencing suppressor gene [4]. Using several model systems, such as cucumber mosaic virus, potyviruses, and tomato bushy stunt virus, the mechanism of these virally encoded silencing suppressors has been elucidated [10, 25,
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Y. Zhai et al. Fig. 1 Alignment of the amino acid sequence of the NSs protein of tomato spotted wilt virus (TSWV) with those of other tospoviruses. (A) Alignment of amino acid sequences of NSs proteins between positions 150–200 and (B) between positions 400–450
37]. In many cases, it has been shown that these silencing suppressors interfere with RNA silencing through doublestranded RNA (dsRNA) binding, which can sequester siRNA duplexes, preventing the small RNA from being loaded into the Argonaut protein (AGO), which is the central catalytic component of RNA-induced silencing complex (RISC), thus interfering with the assembly of the RNA-induced silencing complex [9, 14]. In the case of TSWV, it has been shown that NSs can bind both long and short dsRNAs and interfere with Dicer cleavage [28]. In groundnut bud necrosis virus (GBNV), a tospovirus that is widely prevalent in the Indian subcontinent, the NSs protein has been shown to have NTPase activity and ATP-independent 50 DNA/RNA phosphatase activity [15]. Recently, it has been shown that there is a potential synergistic interaction between iris yellow spot virus and TSWV in doubly infected datura plants, resulting in increased accumulation of NSs of both viruses [2]. The importance of NSs in suppressing the plant defense mechanism underscores the need to gain a better understanding of the structure-function relationships of the NSs gene. However, little is known about the NSs regions that
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are critical in modulating suppressor activity. In the case of tospoviruses, structure-function studies have been hampered by the lack of a reverse genetics system. Most of the studies on identifying genetic determinants of symptom severity or vector transmission, or those assigning functions to various virally encoded genes have utilized naturally occurring or artificially generated virus variants with distinct biological properties. Here, we utilize a widely used ‘reversal-of-gene-silencing assay’ and site-directed mutations to investigate the importance of selected conserved regions of the NSs protein for their relative importance in maintaining suppressor activity. To identify the conserved regions of NSs that may be critical for its suppressor activity, the deduced amino acid sequence of the NSs protein of TSWV was aligned with those of fifteen other tospoviruses (Fig. 1A and B). These included calla lily chlorotic spot virus (CCSV), capsicum chlorosis virus (CaCV), chrysanthemum stem necrosis virus (CSNV), groundnut bud necrosis virus (GBNV), groundnut ringspot virus (GRSV)-African, groundnut ringspot virus (GRSV)-associated-Florida isolate, iris yellow spot virus (IYSV), impatiens necrotic spot virus
Mutational analysis of a tospovirus silencing suppressor
(INSV), melon yellow spot virus (MYSV), polygonum ringspot virus (PolRSV), tomato yellow ring virus (TYRV), tomato zonate spot virus (TZSV), watermelon bud necrosis virus (WBNV), watermelon silver mottle virus (WSMoV), and zucchini lethal chlorosis virus (ZLCV). Two motifs, GKV/T at positions 181-183 and YL at positions 412-413, were found to be conserved among all tospoviruses analyzed. Using a Quikchange Lightning sitedirected mutagenesis kit (Invitrogen, Santa Clara CA), sitedirected point mutations were made to change K 182 to A in the GKT motif (designated as NSs-1) and L 413 to A in the YL motif (designated as NSs-2). Primers for the sitedirected mutagenesis of the GKV motif (A473G_A474C and A473G_A474_antisense) and YL motif (t132g_t133c and t132g_t133c_antisense) were designed using the Agilent QuikChange primer design tool (Supplemental Table 1). The complete NSs gene was amplified by RT-PCR, cloned into cloning vector pART7 [11], and verified by DNA sequencing. The plasmid clone was digested with NotI to release the complete NSs expression cassette containing the CaMV 35S promoter. The NSs expression fragment was then cloned into binary vector pART27 [11] for agroinfiltration. We utilized a ‘‘reversal-of-silencing assay’’ (Agrobacterium co-infiltration assay) to evaluate and assess the silencing suppressor activity of wild-type and mutant constructs of TSWV NSs. The original GFP signal is generated by the GFP gene in transgenic N. benthamiana line 16c. Infiltration of pCAMBIA 1302 can abolish the GFP signal due to co-suppression. Coinfiltration of pCAMBIA1302 and wild-type NSs can suppress RNA silencing, thereby restoring the expression of GFP. Transgenic N. benthamiana line 16c expressing GFP [26], which exhibits green fluorescence under UV light, was used. Non-transgenic N. benthamiana leaves show red fluorescence under UV light due to chlorophyll. The plasmid pCAMBIA1302, harboring 35S:GFP, was used for silencing GFP expression in line 16c. Competent Agrobacterium tumefaciens strain GV3101 cells were electroporated separately with the recombinant plasmid containing the NSs wild type, NSs-1 and NSs-2 constructs. Bacterial cells were then plated on yeast extract-peptone (YEP) plates containing spectinomycin. N. benthamiana line 16c leaves were agroinfiltrated at the 5- to 6-leaf stage. A. tumefaciens was inoculated in 5 ml of YEP liquid medium containing spectinomycin and rifampicin and incubated for 20 h at 28 °C with vigorous shaking. A 2-ml culture was transferred to fresh 50 ml YEP liquid medium containing spectinomycin and rifampicin and grown for 20 h at 28 °C with vigorous shaking. A. tumefaciens cultures were centrifuged at 4000 rpm for 10 minutes, and the cells were resuspended in infiltration buffer (10 mM
MgCl2, 10 mM MES, pH 5.7, 2.25 mM acetosyringone). The volume of the culture was adjusted with the infiltration medium to a final optical density (OD) of 0.5 at 600 nm. Equal volumes of each A. tumefaciens culture were mixed. Before infiltration, the mixture was incubated in the dark without shaking for six hours [16]. After infiltration, the plants were maintained in a greenhouse (16 hours of light and eight hours of darkness). GFP fluorescence was detected using a 365-nm long-wave UV lamp. Leaves were photographed using a Panasonic Lumix digital camera. Leaves of GFP-expressing N. benthamiana line 16c agroinfiltrated with pCAMBIA 1302 alone do not exhibit GFP fluorescence due to co-suppression of GFP gene expression. However, co-infiltration of pCAMBIA1302 and the wild-type TSWV NSs construct restores GFP fluorescence due to the silencing suppressor activity of TSWV NSs. In the case of NSs-1 and NSs-2, in which selected amino acids were mutated, if these mutations affected the suppressor activity of NSs, restoration of GFP fluorescence would be expected to be diminished or completely abolished in leaves co-agroinfiltrated with either of these constructs along with pCAMBIA1302. The line 16c leaves co-agroinfiltrated with pCAMBIA1302 and the wild-type NSs construct showed expression of GFP under UV light, indicating silencing suppressor activity of TSWV NSs (Fig. 2A). Line 16c leaves infiltrated with pCAMBIA1302 alone as a negative control exhibited red fluorescence under UV light, as expected (Fig. 2B). The line 16c leaves co-agroinfiltrated with pCAMBIA1302 and TSWV NSs-1 or NSs-2 mutant did not show any GFP expression (Fig. 2C, D) indicating that the ability of NSs-1 to act as a suppressor was negatively affected as a consequence of these mutations. A double mutant consisting of NSs-1 and NSs-2 was made (referred to as NSs-3) and tested for its effect on NSs suppressor activity. When coagroinfiltrated with pCAMBIA1302, the NSs-3 mutant also failed to restore GFP expression, and only red fluorescence was observed (Fig. 2E). To confirm that the amino acid substitutions did not have any influence on the stability of the NSs protein, a direct antigen-coated-ELISA (DAC-ELISA) assay was performed on all the co-agroinfiltrated leaves using a monoclonal antibody that is specific for the TSWV NSs (Agdia, Elkhart IN). The N. benthamiana samples were ground in a 1:1 (w/v) dilution in carbonate buffer containing 2 % polyvinyl pyrrolidone (PVP, MW 40,000). The TSWV NSs antiserum was diluted (1:5000), and antimouse immunoglobulin alkaline phosphatase (universal conjugate, Sigma, USA) was diluted (1:20,000) in PBSTPO buffer. Absorbance values at 405 nm were recorded using a, BioTek ELX 800 microplate reader. TSWVinfected leaves were used as a positive control. The NSs protein could be detected by ELISA in leaves co-
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Fig. 2 Effect of point mutations on the silencing suppression activity of tomato spotted wilt virus (TSWV) NSs in Nicotiana benthamiana line 16c plants. A, leaf from N. benthamiana line 16c plants infiltrated with a mixture of pCAMBIA1302 and full-length TSWV NSs in a p7/ p27 construct. B, leaf from N. benthamiana line 16c plants infiltrated with pCAMBIA1302 and empty p7/p27 vector. C, leaf from N. benthamiana line 16c plants infiltrated with a mixture of pCAMBIA1302 and the NSs-1 construct (GKV motif mutant). D, leaf from N. benthamiana line 16c plants infiltrated with mixture of pCAMBIA1302 and the NSs-2 construct (YL motif mutant). E, leaf from N. benthamiana line 16c plants infiltrated with a mixture of
pCAMBIA1302 and double mutant (mutations in both the GKV and YL motifs). F, western blot analysis for NSs detection of three mutant constructs. Lane 1, N. benthamiana line 16c leaf infiltrated with empty p7/p27. Lane 2, TSWV-infected line 16c leaf as a positive control. Lane 3, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and the TSWV NSs full sequence (wild-type NSs) in a p7/ p27 construct. Lane 4, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and the NSs-1 construct. Lane 5, line 16c leaf infiltrated with a mixture of pCAMBIA1302 and NSs-2 construct. Lane 6, line 16c leaf infiltrated with mixture of pCAMBIA1302 and NSs-3
agroinfiltrated with either the wild-type TSWV NSs or those with mutant constructs (data not shown). Western blotting was performed to verify the results of ELISA. The antisera used consisted of TSWV NSs-specific monoclonal antibody (Agdia Inc., Elkhart, IN) as the primary antibody (used at 1:5000 dilution) and anti-mouse immunoglobulin conjugated with alkaline phosphatase (Sigma; used at 1:20,000 dilution) as the secondary antibody. Protein bands were visualized using BCIP/NBT as substrates. The NSs protein could be detected in leaves agroinfiltrated with all three mutant constructs, confirming that the mutations did not affect the expression or stability of the protein (Fig. 2F). It has been shown that almost all plant viruses code for a suppressor of gene silencing [4]. Virally encoded suppressors act by interacting with dsRNA and interfering with the assembly of the RISC [14]. In the case of tospoviruses, it has
been shown that the NSs protein acts as the silencing suppressor [32] and is an avirulence determinant [19]. Among several possible mechanisms, it can interact with siRNA or dsRNA and prevent them from loading to the RISC [28]. A recent report suggested that the NSs of GBNV contains NTPase activity and ATP-independent 50 DNA/RNA phosphatase activity. The mutation of K189 to A in the GKT motif resulted in the complete loss of ATPase activity. However, the 50 phosphatase activity was not affected by the mutation [15]. Our results indicated that the conserved amino acids in TSWV NSs play a vital role in its suppressor function. Both N-terminal and C-terminal sequences are indispensable for the silencing suppressor function of the NSs protein. In our study, two constructs were made, each with either the N part (1-227 aa) or C part (245-467 aa) of the protein. Neither of the truncated proteins retained the suppressor activity (Fig. 3).
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Mutational analysis of a tospovirus silencing suppressor
Fig. 3 Effect of deletions in the NSs gene of tomato spotted wilt virus (TSWV) on its gene suppressor activity in Nicotiana benthamiana line 16c plants. A, leaf infiltrated with a mixture of pCAMBIA1302 and a full-length gene TSWV NSs sequence in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). B, leaf infiltrated with a mixture of pCAMBIA1302 and the truncated
gene containing the amino-terminal 227 aa of TSWV-NSs in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). C, leaf infiltrated with a mixture of pCAMBIA1302 and the carboxyl terminal aa 245-467 of TSWV-NSs in a p7/p27 construct (a), and a leaf infiltrated with pCAMBIA1302 (b). Arrows indicate the infiltrated area on the leaf
Amino acids that are crucial for protein function or binding capacity have been identified in tospovirus NSs proteins. For example, Cheng et al. [5] identified nine core amino acids from watermelon silver mottle virus (WSMoV) that interact with NSscon (23 aa; a common epitope in the gene silencing suppressor NSs proteins of the members of the WSMoV serogroup) monoclonal antibody. In our study, three conserved motifs were identified in the TSWV NSs protein: GKV (aa 181-183), KTL (aa 398-400), and YL (aa 412-413). In order to identify the functional motifs to which these conserved sites belong, the NSs protein sequence was scanned using protein function site prediction software (http://elm.eu.org/). The KTL region does not exist in any of the identified motifs and was not included in this investigation. The GKV region is within the motif recognized by the Src Homology 3 (SH3) domain, and the YL region is within the Src Homology 2 (SH2) domain binding motif (Supplemental Table 2). Past reports have revealed the importance of SH2 domain–SH2 binding motif and SH3 domain–SH3 binding motif interactions in virus replication and signal transduction pathways [6, 17, 23]. In one recent example from hepatitis C virus (HCV) [24], the Src family kinase c-Src was shown to interact directly with viral RdRp via its SH3 domain, and with nonstructural phosphoprotein NS5A via its SH2 domain. The SH2 and SH3 domains are essential for the interaction between c-Src and RNA-binding proteins [31] and the regulation of their RNA-binding ability [8]. Mutations in the GKV or YL regions caused the disruption of SH2- and SH3-binding motifs in NSs, which probably affected its RNA-binding ability and therefore suppressed its anti-RNAi function. Our findings provide the first experimental evidence for the role of selected regions that are critical for TSWV NSs suppressor activity. Information about the relative
importance of these conserved motifs could be useful in developing novel RNAi-based approaches for antiviral strategies. Acknowledgements This work was supported by the WSU Agricultural Research Center, PPNS No. 0627, Department of Plant Pathology, College of Agricultural, Human and Natural Resource Sciences, Agricultural Research Center, Project # WNPO 0545, Washington State University, Pullman, WA, 99164-6430, USA. HRP’s visit to Instituto Virologia Vegetale, CNR, Turin, Italy, was supported by a fellowship from the OECD under the Cooperative Research Program.
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