The RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD).
Methods
CLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0’s silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves.
Results
High sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery.
Conclusions
The sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.
The online version of this article (doi:10.1186/s12985-015-0356-7) contains supplementary material, which is available to authorized users.
Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
RSC, GSM, MFSV and RLC conceived and designed the experiments. RSC, ILGA and TFS performed the experiments. MFSV and TFS contributed with plant material. GSM, MFSV and RLC contributed reagents/materials/analysis tools. RSC and RLC wrote the manuscript. All authors read and approved the final manuscript.
Background
Cotton leafroll dwarf virus (CLRDV) is the causal agent of an economically important cotton (Gossypium hirsutum) disease called cotton blue disease (CBD) [1]. Aphis gossypii-transmitted CBD has been observed in several cotton-producing areas of Central Africa, Asia and South America [2]. CBD symptoms are characterized by stunting, leaf rolling, vein yellowing, dark-green leaves and small bolls, leading therefore to severe yield losses when aphid populations are not properly controlled. In Brazil, CBD is present in almost all cotton growing fields and the disease was also partially controlled by the application of insecticides to decrease aphid populations and by the use of CBD-resistant cotton cultivars. Since 2006, several resistance breaking CLRDV isolates have been observed throughout the country, producing CBD-like symptoms in formerly resistance cotton lines [3]. Apart from typical CBD symptoms, resistant or susceptible cotton varieties infected with CLRDV resistance-breaking isolates may also display reddish and withered leaves. Resistance breaking isolates are now widely distributed in Brazilian cotton growing areas, making the use of insecticide for aphid control compulsory.
The CLRDV genome resembles a typical member of the genus Polerovirus, family Luteoviridae and contains six open reading frames (ORF0 to ORF5) [4]. The genome is divided into two gene-containing portions, separated by an approximately 200 nucleotides intergenic region. Three open reading frames (ORF3, ORF4 and ORF5) are located in the 3’-end portion of the genome encoding for the structural proteins (capsid, movement and aphid-transmission proteins, respectively), while the 5’-end region of the genome encodes replication-related proteins (ORF1 and ORF2) and also a gene (ORF0) encoding the RNA silencing suppression protein P0. In general, the genome sequences of resistance breaking CLRDV isolates are very similar to CLRDV isolates from susceptible plants [3]. For example, the degree of sequence identity in all proteins encoded by ORFs 1 to 5 is greater than 93 % between two resistance breaking CLRDV isolates (Ima2 and Acr3) and two non-resistance breaking ones (PV1 and ARG) in cotton plants. However, when the identities among P0 proteins are compared, the diversity is consistently higher, with identity numbers ranging from 85.8 to 86.6 % among the four isolates [3].
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The P0 protein from several members of the genera Polerovirus and Enamovirus, family Luteoviridae, are known to be involved in the suppression of plant’s anti-viral defense mechanisms at variable degrees, depending on the species and isolates [5‐11]. P0’s silencing suppression activity is mediated by promoting the destabilization of ARGONAUTE (AGO) proteins, key players in RNA silencing mechanisms [8, 12‐15]. In plants, the RNA silencing pathway is triggered by double stranded RNAs (dsRNAs), which are processed by Dicer-like enzymes into small RNAs ranging from 20 to 24 nucleotides [16]. Viral-derived small interfering RNAs (siRNAs) produced during infections are readily recruited by AGO-containing RNA-induced silencing complexes (RISC) and used by the machinery to degrade viral genomic and sub-genomic sequences, being therefore an efficient anti-viral defense mechanism [17]. AGO is an important component of the machinery, since it directly binds to siRNAs and guide RISC to target RNAs. Viral RNA degradation may take place either at locally infected cells or at distal tissues, by the systemic movement of silencing signals [18]. A plethora of evolutionary unrelated viral proteins has evolved to cope with the anti-viral RNA silencing process. The P19 proteins from tombusviruses are one of the best characterized suppressors. P19 proteins are able to bind to sRNAs, preventing their loading into RISC [19]. Similarly, by degrading AGO proteins, P0 proteins are able to suppress the plant’s anti-viral defense, allowing the infection to proceed. P0 proteins probably exert their activity through an F-box-dependent interaction with homologs of the S-phase kinase-related protein 1 (SKP1) ASK1 and ASK2 [20]. SKP1 is a core component of the SKP1/Cullin1/F-box (SCF) family of E3 ubiquitin ligases that mediate the ubiquitination of diverse regulatory and signaling proteins [21]. Point mutations in P0’s F-box motif may abolish its interaction with SKP1 and consequentially decreasing AGO destabilization and viral pathogenesis [8, 9, 11, 20]. However, P0’s activity is insensitive to proteasome inhibitors and the viral protein probably operates by hitchhiking cellular autophagy pathways endogenously used to modulate AGO homeostasis [13‐15]. This model is supported by the increased accumulation of AGO proteins in the presence of autophagy inhibitors and by its co-localization with autophagic vesicles [14].
Recently, the P0 protein from an Argentinian isolate of CLRDV (P0CL-ARG) has been characterized as a RNA silencing suppression protein [10]. The level of both local and systemic silencing suppression observed in P0CL-ARG seems to be low when compared to other members of the group. Almost no suppression of systemic silencing is observed for P0CL-ARG, in line with what has been previously found for the P0 proteins from other members of the family [5, 10, 22]. However, it’s known that P0 silencing suppression activity can vary even among closely related viruses. For example, European isolates of beet mild yellowing virus vary greatly in their ability to suppress local silencing [7]. Here, the local and systemic silencing suppression activities of P0 proteins from CLRDV isolates collected in different parts of Brazil, including CBD resistant and susceptible cotton varieties, were assessed and compared to P0CL-ARG. Results indicate that silencing suppression capabilities are strain-specific and that strength of local and systemic silencing suppression is not correlated in CLRDV P0 proteins.
Results and discussion
Sequence diversity among CLRDV P0 proteins
When the genomic sequences of four previously identified CLRDV isolates were compared, significant differences were only observed in the P0 coding sequence [3]. In order to better characterize the sequence diversity of P0 proteins among CLRDV isolates, cotton plants from different varieties, displaying typical or atypical CBD symptoms were collected from different parts of Brazil. Sampled areas covered the main cotton producing areas of the country, most of them with significant geographical distance from each other (Fig. 1). In total, seven Brazilian CLRDV isolates (PV1, Ima2, Acr9, Ipa4, Pm1, Hol1 and Pal3) were analyzed and compared to the Argentinian isolate (ARG) [4] and also to the P0 from an Australian isolate of potato leafroll virus (P0PL-AU) [8] (Table 1). The genomes from two of the Brazilian isolates analyzed (PV1 and Ima2) have been previously obtained [3, 23]. Three CLRDV isolates (Acr9, Ima2 and Ipa4) were obtained from known cotton CLRDV-resistant varieties and are therefore treated as resistance-breaking isolates. Two isolates (Ima2 and Pm1) were obtained from plants showing atypical symptoms (Table 1).
Fig. 1
Map of Brazil showing sites where cotton plants were harvested for the study. The numbered red dots in the map indicate the different cotton leafroll dwarf virus isolates harvested
Table 1
Brazilian isolates of cotton leafroll dwarf virus used in the study
Isolate
Locationa
G. hirsutum cultivar
CBD resistance phenotype
Symptoms observed
Year
PV1
Primavera do Leste – MT
FM966
Susceptible
Typical
2004
Acr9
Acreuna – GO
CD406
Resistant
Typical
2006
Hol1
Holambra – SP
Nd
Nd
Typical
2007
Ima2
Campo Verde - MT
IAC25 RMD
Resistant
Atypical
2009
Ipa4
Ipameri - GO
Delta Opal
Resistant
Typical
2006
Pal3
Palotina - PR
CD034928
Nd
Typical
2006
Pm1
Patos de Minas - MG
Epamig1
Nd
Atypical
2007
aMT, Mato Grosso State; GO – Goiás State; SP – São Paulo State; PR – Paraná State; MG – Minas Gerais State
×
The amino acid sequence identity among CLRDV P0s varied from 85.5 % (P0CL-Ima2/P0CL-Hol1) to 98.88 % (P0CL-Pm1/P0CL-Pal3 and P0CL-ARG/P0CL-Pal3) (Table 2). The P0 sequences from the isolates Hol1, Pal3 and Pm1 are very close to the P0s from PV1 and ARG, the two isolates initially associated with CBD [1, 4], with identities ranging from 97.76 % to 98.88 % (Table 2). The isolates Acr9, Ima2 and Ipa4 also have a high sequence identity among them, ranging from 95.53 % to 97.2 %. The identity among P0 sequences from isolates Acr9, Ima2 and Ipa4, however, is lower than 90 % when compared to PV1 or ARG. The current taxonomic criteria in the family Luteoviridae states that viruses having amino acid divergence higher than 10 % in any protein sequence should be considered as different species [24]. In this line, the isolates Acr9, Ima2 and Ipa4 should be regarded as a new species associated with CBD. Since P0 sequences are the most variable sequences among poleroviruses, it has been recently proposed that viruses having high diversity in this region, but with amino acid identities higher than 90 % in all other proteins should be regarded as genotypes of the same species and not as a different one [25, 26]. This kind of analysis can only be made for the Ima2 isolate, the only one of the three with the genome fully sequenced [3]. But since the P0 sequences of the three isolates are very similar (Table 2), Acr9 and Ipa4 are probably also new genotypes of CLRDV, as previously stated for Ima2 [3].
Table 2
Percentage of amino acid identity among the viral isolates used in the study
P0CL-Pal3
X
P0CL-Pm1
98.88
X
P0CL-PV1
98.51
98.14
X
P0CL-Hol1
98.51
98.14
97.76
X
P0CL-ARG
98.88
98.51
98.14
98.14
X
P0CL-Ima2
86.24
85.87
86.61
85.5
86.24
X
P0CL-Ipa4
88.1
87.73
87.73
87.36
87.36
96.28
X
P0CL-Acr9
88.1
87.73
87.73
87.36
88.1
95.53
97.02
X
P0PL-AU
18.21
18.21
17.84
18.21
18.58
17.84
18.58
18.58
X
P0CL-Pal3
P0CL-Pm1
P0CL-PV1
P0CL-Hol1
P0CL-ARG
P0CL-Ima2
P0CL-Ipa4
P0CL-Acr9
P0PL-AU
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The separation of the isolates in two groups is also supported by phylogenetic analysis. It has been shown that, when full genomic sequences are not available, P0-based phylogenies can correctly reconstruct the relatedness among poleroviruses [25]. A Neighbor-joining tree based on CLRDV P0s groups the isolates Hol1, Pal3 and Pm1 with the two CBD founding members PV1 and ARG, while the isolates Acr9, Ima2 and Ipa4 clearly branch out, forming a well-supported group (Fig. 2). Since the three divergent isolates were all obtained from formerly cotton resistant varieties (Table 1), the silencing suppression activities from all isolates were then scored and compared.
Fig. 2
Neighbor joining phylogenetic tree of the cotton leafroll dwarf virus isolates used in the study. Tree was constructed based on P0’s amino acid sequence alignments. Numbers above the lines indicate the bootstrap scores with 1,000 replicates. Scale bar represents genetic distance. The Australian isolate of PLRV P0 was used as outgroup
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Suppression of local silencing by CLRDV P0s
Previous data have shown that P0CL-ARG is weak suppressor of local silencing when expressed in Nicotiana benthamiana leaves [10]. The suppression assay is based on the Agrobacterium-mediated transient co-expression of mGFP5 and a candidate silencing suppression protein in mGFP5-expressing N. benthamiana leaves (transgenic line 16c) [27]. When no silencing suppression is observed, the transiently expressed GFP triggers a strong RNA silencing response that ultimately leads to RNA degradation from both stable and transient transgenes. However, in the presence of a silencing suppression protein, GFP degradation is prevented and both transgenes (stable and transient) are expressed, increasing total GFP levels. The silencing suppression assay can be easily monitored by hand UV lamps. In order to check whether the sequence diversity observed among the P0s could also reflect variable silencing suppression activities, the P0 from all seven Brazilian isolates and the Argentinian isolate were tested side-by-side in the 16c N. benthamiana line and compared to P0PL-AU and P19, two known suppressors of local silencing. All genes were cloned into the pGWB417 binary vector [28], leading to a 35S-driven expression of Myc-tagged proteins.
As expected, a red patch in the infiltrated area was observed when GFP was expressed in the absence of a silencing suppression protein (Fig. 3). Conversely, strong GFP fluorescence was observed in the presence of P19 or P0PL-AU in all time-points analyzed (Fig. 3). All tested CLRDV P0s displayed obvious RNA silencing suppression activities. When scored based on GFP fluorescence, the levels of silencing suppression activity observed for the CLRDV P0s were similar to P0PL-AU, but significantly lower than the P19 suppressor protein even at 3 days post-infiltration (dpi) (Fig. 3). The observed GFP fluorescence at 3 dpi correlates well with the accumulation of GFP RNAs when checked by real-time PCR at this time-point in N. benthamiana 16c plants (Fig. 4a). GFP RNA levels in GFP/P19-infiltrated plants were approximately 14 times higher than in control mock-infiltrated 16c plants. However, the GFP RNA fold change in P0PL-AU or CLRDV P0-infiltrated plants, varied from only 2 to 6 times the levels obtained in the same control condition (Fig. 4a). At later time-points, GFP fluorescence started to fade at infiltrated areas of P0CL-Acr9, P0CL-Hol1, P0CL-Ima2 and P0CL-Ipa4, indicating that those proteins are weaker suppressors than the other P0s (Fig. 3). The accumulation of the Myc-tagged suppressor proteins in the three biological replicates used for real-time PCR was checked by western blot using anti-Myc antibodies (Fig. 4b). Although all suppressors were expressed from the same vector background (pGWB417) [28], the P19 protein accumulated at levels consistently higher than the P0s. The strong suppression activity observed for P19 in the assay, therefore, might be correlated with its higher stability in N. benthamiana leaves. Since the P0s accumulated at similar levels, the observed differences in silencing suppression activities for those proteins might be due to functional divergence and not expression levels.
Fig. 3
Suppression of local silencing by cotton leafroll dwarf virus P0s in N. benthamiana 16c plants. Transgenic N. benthamiana plants expressing GFP (line 16c) were co-infiltrated with Agrobacterium carrying plasmids to express GFP (silencing trigger) and candidate suppressor proteins (P0CL-PV1, P0CL-Acr9, P0CL-Hol1, P0CL-Ima2, P0CL-Ipa4, P0CL-Pal3 and P0CL-Pm1). GFP co-expressed with empty vector was used as negative control. P0CL-ARG, P0PL-AU and P19 were used as positive controls in the assay. Pictures were taken at 3, 6 and 9 days post-infiltration (dpi)
Fig. 4
Accumulation of GFP mRNA and suppressor proteins in infiltrated N. benthamiana 16c leaves at 3 days after infiltration (dpi). a GFP levels in tissues co-infiltrated with cotton leafroll dwarf virus P0 proteins (P0CL-ARG, P0CL-PV1, P0CL-Acr9, P0CL-Hol1, P0CL-Ima2, P0CL-Ipa4, P0CL-Pal3 or P0CL-Pm1), potato leafroll virus P0 (P0PL-AU), empty vector or mock-inoculated were detected with real-time PCR. Error bars indicate standard deviation of GFP mRNA in three biological repeats. Normalized value obtained in the mock sample was arbitrary set to 1 and all the other values compared to it. Data was normalized with Ubi3 and EF-1 reference genes. Asterisks indicate values that are statistically different from the control mock construct, with p-values varying from 0.0002 to 0.0478. b Western blot showing the accumulation of suppressor proteins. Protein extracts from all the three biological replicates from each construct used for real-time PCR were run in SDS-PAGE, transferred to membranes and probed with Myc-tag specific antibodies. Gel loading was observed by Ponceau staining. Non-infiltrated plants (NI) were used as negative controls. Top left panel: accumulation of the control proteins P0PL-AU and tombusvirus P19 and the negative control (infiltrated with the empty vector). Top right panel: accumulation of P0CL-ARG, P0CL-PV1 and P0CL-Acr9 proteins. Bottom left panel: accumulation of P0CL-Hol1, P0CL-Ima2 and P0CL-Ipa4 proteins. Bottom right: accumulation of P0CL-Pal3 and P0CL-Pm1 proteins
×
×
The fading phenotypes observed in 16c plants for the suppressor proteins P0CL-Acr9, P0CL-Hol1, P0CL-Ima2 and P0CL-Ipa4 were reproduced when the experiment was repeated in wild type plants (Additional file 1: Figure S1). When transiently expressed alone in wild type plants, the accumulation of GFP was lower than in the presence of the control strong suppressor P19, even at 3 dpi (Additional file 1: Figure S1). At 6 dpi, GFP was almost totally silenced when expressed alone, contrasting to what was observed in the presence of control constructs (P19 or PLP0-AU) or any of the CLRDV P0s. From 6 dpi onwards, as mentioned before, GFP levels started to fade in the presence of P0CL-Acr9, P0CL-Hol1, P0CL-Ima2 and P0CL-Ipa4, also indicating that those proteins are not able to suppress GFP silencing in N. benthamiana leaves for long periods. In all time-points analyzed, GFP accumulated at levels expressively higher when co-infiltrated with P19 than in the presence of any other P0, indicating that even the ones able to maintain GFP suppression at later times post-infiltration (P0PL-AU, P0CL-ARG, P0CL-PV1, P0CL-Pal3 and P0CL-Pm1) should be regarded as moderate suppressors compared to the control used in the assays.
It has been previously shown that P0s depend on the presence of a F-box-like motif to exert their silencing suppression activity [8, 9, 11, 20]. The hallmark amino acids LPxx(L/I)x10–13P could be found in all CLRDV P0s tested (Additional file 2: Figure S2). However, isoleucine is changed to an amino acid with similar biochemical properties (valine) in the three resistance-breaking CLRDV isolates (Acr9, Ima2 and Ipa4). The ring structure amino acids known to affect local silencing suppression activity of melon aphid-borne yellows virus P0 are also present and conserved among the CLRDV P0s (Additional file 2: Figure S2) [9]. Therefore, the local silencing suppression variability observed among CLRDV P0s is probably associated with alternative functional residues.
Suppression of systemic silencing by CLRDV P0s
Viral siRNAs produced in infected cells may also move systemically through vascular tissues to reach other parts of the plants [18]. In the N. benthamiana 16c assay, systemic silencing can be visualized by the appearance of red-silenced areas especially around the veins of newly developed leaves. Eventually, silencing signals may spread throughout the leaves, producing completely silenced plants. The systemic silencing suppression activities of all CLRDV P0s were scored and compared to the strong systemic suppressor P0PL-AU [8]. At 16 dpi, 15 out of 20 plants assayed showed systemic silencing when infiltrated with the GFP silencing-trigger construct in the absence of any suppressor protein and almost 100 % of the plants were silenced by 20 dpi (Table 3). As expected, P0PL-AU completely blocked the spread of silencing signals as no silenced plants were observed at 16, 20 or 29 dpi. However, the suppression of systemic silencing mediated by the CLRDV P0s varied among the different isolates. In our experimental conditions, only 3 plants out of 20 were silenced at 16 dpi when co-infiltrated with P0CL-ARG (Table 3). The number of silenced plants, however, increased to 7 and 10 out of 20 at 20 dpi and 29 dpi, respectively, indicating that P0CL-ARG is a moderate suppressor of silencing signals (Table 3). This result contrast to what has been previously observed for P0CL-ARG, where 8 out 10 plants were already silenced by 15 dpi in the presence of the protein [10]. Since the spread of RNA silencing signals may be influenced by environmental conditions [29‐31] and possibly by the number of infiltrated leaves, concentration and strain of Agrobacterium used in the assay and vector background, the difference in the systemic silencing activity observed for P0CL-ARG might be due to different experimental settings.
Table 3
Proportion of plants showing systemic silencing at 16, 20 and 29 days post-infiltration (dpi)
Infiltration
16 dpi
20 dpi
29 dpi
GFP + Vector
15/20
19/20
19/20
GFP + P0PL-AU
0/20
0/20
0/20
GFP + P0CL-PV1
0/20
0/20
0/20
GFP + P0CL-ARG
3/20
7/20
10/20
GFP + P0CL-Acr9
5/20
7/20
7/20
GFP + P0CL-Hol1
0/20
1/20
2/20
GFP + P0CL-Ima2
7/20
9/20
11/20
GFP + P0CL-Ipa4
0/20
0/20
0/20
GFP + P0CL-Pal3
0/20
0/20
0/20
GFP + P0CL-Pm1
1/20
1/20
1/20
In line with what has been observed for P0CL-ARG, the P0 proteins P0CL-Acr9 and P0CL-Ima2 were also moderate suppressors of systemic silencing (Table 3). However, the P0 proteins from five isolates (PV1, Hol1, Ipa4, Pal3 and Pm1) efficiently blocked the spread of systemic silencing signals, with a silencing suppression activity similar to P0PL-AU (Table 3). During the experiments, 70 % of the plants infiltrated with suppressor proteins P0CL-Pal3 and P0CL-Pm1 and 50 % of P0PL-AU-infiltrated ones displayed strong necrotic lesions at late times after infiltration, most of them starting at 10 dpi (data not shown). Therefore, the suppression of systemic silencing by those proteins could have also been influenced by the induced cell death. Similar necrotic phenotypes have also been observed for P0PL-AU [8] and for the P0s from sugarcane yellow leaf virus and beet western yellows virus [6].
AGO destabilization by CLRDV P0s
The P0 proteins from some members of the family Luteoviridae are able to destabilize the expression of AGO proteins [8, 11‐15]. However, the activity of CLRDV P0 in AGO decay has never been tested. For that, a Myc-tagged version of the Arabidopsis thaliana AGO1 protein, known to be involved in several RNA silencing pathways, including anti-viral defense [32, 33], was transiently expressed via Agrobacterium infiltration in wild type N. benthamiana leaves in the presence or absence of different P0s or control constructs. The accumulation of AtAGO1-Myc protein was detected by anti-myc antibodies when co-expressed without P0 suppressors or in the presence of P19 (Fig. 5). All P0s, including P0PL-AU, the seven Brazilian isolates and the Argentinian isolate of CLRDV were able to strongly decrease the AtAGO1-Myc levels when co-expressed. Therefore, the local and systemic silencing differences observed among the isolates might be due to the regulation of still unknown cellular proteins by P0, possibly other AGO members [8], or due to small differences in AGO1 accumulation that are not detected due to western blot resolution limits.
Fig. 5
Accumulation of a Myc-tagged version of the A. thaliana AGO1 protein transiently expressed in N. benthamiana leaves in the presence or absence of P0 proteins. Myc-tagged versions of cotton leafroll dwarf virus P0s (P0CL-ARG, P0CL-PV1, P0CL-Acr9, P0CL-Hol1, P0CL-Ima2, P0CL-Ipa4, P0CL-Pal3 or P0CL-Pm1) and potato leafroll virus P0 (P0PL-AU) were used in the assay. Non-infiltrated plants (NI) were used as negative controls and AtAGO1 infiltrated with the empty vector or with a Myc-tagged P19 were used as positive controls. Membranes were probed with anti-Myc antibodies. Ponceau staining was used as a loading control. All co-infiltrations were performed in the presence of P19 (without tag) to stabilize mRNA Agrobacterium-mediated experiments
×
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Conclusions
Our results indicated a high diversity among P0 proteins from Brazilian and Argentinian isolates of CLRDV, a virus associated with CBD. All CLRDV P0 proteins analyzed were able to mediate AtAGO1 decay, however, variable silencing suppression activities were observed, probably reflecting their sequence diversity. P0CL-ARG was a moderate silencing suppressor of both local and systemic silencing in our experiments, when compared to the positive control constructs used in the assays (Figs. 3, 4 and Additional file 1: Figure S1). Three proteins (P0CL-PV1, P0CL-Pal3 and P0CL-Pm1) were also moderate suppressors of local silencing, but strong suppressors of systemic silencing. Four other proteins behaved as weak suppressors of local silencing. Contrasting to control constructs (P19 and P0PL-AU) and to other CLRDV P0s, those four proteins (P0CL-Acr9, P0CL-Ima2, P0CL-Hol1 and P0CL-Ipa4) could not support GFP suppression for long periods when assayed in the mGFP5-expressing N. benthamiana 16c line (Fig. 3) or in wild type plants (Additional file 1: Figure S1). GFP levels clearly started to fade in the presence of those proteins from 6 dpi onwards (Fig. 3 and Additional file 1: Figure S1). However, two of the weak local silencing suppressor proteins (P0CL-Hol1 and P0CL-Ipa4) were able to almost completely block the spread of systemic silencing signals when assayed in 16c transgenic lines. Despite of their weak local silencing, P0CL-Hol1 and P0CL-Ipa4 are as strong as the control P0PL-AU protein in suppressing systemic silencing. It’s tempting to speculate therefore that the strength of local and systemic silencing suppression activity might be genetically unlinked in P0 proteins. Furthermore, these data indicate that the silencing suppression capabilities of the distinct CLRDV P0 proteins are not directly linked to their genetic diversity.
Methods
Plant material and DNA constructs
Gossypium hirsutum plants belonging to at least six cultivars (FM966, CD406, CD034928, IAC25 RMD, Delta Opal and Epamig1) were collected in five different States of Brazil (Goiás, Mato Grosso, Minas Gerais, Paraná and São Paulo) (Table 1 and Fig. 1). The Ima2 isolate was collected in Campo Verde – Mato Grosso, but passed through the IAC24 RMD cotton variety at Instituto Matogrossense do Algodão (Primavera do Leste – Mato Grosso) before being sent for analysis [34]. Harvesting and maintenance of plants were performed according to Brazilian rules (MP 2.186-16/2001). Total RNA of all plants were extracted using Qiagen Plant RNA kit and 2.5 μg were used to prepare cDNAs with the O5R2 primer (5’-GCAACCTTTTATAGTCTCTCCAAT-3’), which anneals in the middle of CLRDV ORF5. The ORF0 sequences from all Brazilian isolates were amplified with primers CLP0_F (5’-CACCATGTTGAATTTGATCATCTGCAG-3’) and CLP0_R (5’-ACTGCTTTCTCCTTCAC-3’) and cloned into pENTRY-D-TOPO (Invitrogen). The ORF0 of the Argentinian isolate of CLRDV [GenBank: NC_014545.1] was synthesized and cloned into a pUC plasmid by the Blue Heron Biotechnology Inc (USA). The Argentinian ORF0 [10] and the P19 coding sequence [35] were amplified with primers CLP0_TOPO_F/CLP0_R and P19_TP_F (5’-CACCATGGAACGAGCTATACAAGGAAACG -3’)/P19_R (5’-TTACTCGCTTTCTTTTTCGAAGG-3’), respectively, and also cloned into pENTRY-D-TOPO (Invitrogen). All amplifications were performed with the Phusion High Fidelity Polymerase (NEB). Entry vectors containing the Arabidopsis thaliana Ago1 coding sequence [TAIR: AT1G48410] and the P0 from the Australian isolate of PLRV [GenBank: D13953.1] were described previously [8].
Genes in entry gateway clones were sequenced in both directions in automated ABI sequencers through dye terminator cycle method, using primers annealing in vector sequences. The accession numbers for the new P0 sequences obtained here are: [GenBank:KR185733] (isolate Acr9), [GenBank:KR185734] (isolate Pm1), [GenBank:KR185735] (isolate Hol1), [GenBank:KR185736] (isolate Ipa4), [GenBank:KR185737] (isolate Pal3). All genes in entry vectors were transferred through LR reactions to the binary destination vector pGWB417 [28], resulting in 35S-driven, Myc-tagged proteins when expressed in plants.
Sequence analysis
Multiple sequence alignments of deduced amino acid sequences were performed with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and phylogenetic reconstructions were performed with the MEGA 4 software [36]. Trees were constructed by the neighbor-joining (NJ) method [37], with the pair-wise deletion option and number of differences matrix.
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Agroinfiltration
Agrobacterium tumefaciens, strain GV3101, were infiltrated in Nicotiana benthamiana leaves as described previously [38]. Cells were individually diluted to an optical density of 1.0 at 600 nm before mixing the cultures. Leaves were infiltrated in the abaxial surfaces with needleless syringes and the infiltrated plants were incubated in growth chambers with a 16-hour photoperiod at 24 °C.
For the local silencing suppression assay, three leaves of 5-weeks-old mGFP5-expressing N. benthamiana plants (wild type or 16c line) [27] were co-infiltrated with equal volumes of A. tumefaciens harboring plasmids expressing mGFP5 and pGWB417 with or without candidate silencing suppressor genes. For the systemic silencing suppression assay, only one leaf of 3-weeks-old N. benthamiana 16c plants were co-infiltrated with equal volumes of A. tumefaciens expressing mGFP5 and pGWB417 (negative control) or with pGWB417 expressing candidate silencing suppressor genes. GFP fluorescence was observed under a long-wavelength UV lamp and the number of plants having systemic silencing scored in different time-points.
For the AGO1 destabilization assay, three leaves of 5-week-old wild type N. benthamiana plants were infiltrated with A. tumefaciens harboring plasmids pJL3:P19 [35], pGWB417-AtAGO1-MYC, and pGWB417 with or without candidate silencing suppression genes in a proportion of 30 %, 35 % and 35 %, respectively. The infiltrated leaves were collected 4 days after infiltration.
qRT-PCR
Total RNA from approximately 100 mg of infiltrated N. benthamiana leaf tissue was extracted using the Plant RNA Purification Reagent (Invitrogen) according to manufacturer’s instructions. The quality of the RNA was checked by electrophoresis on 1.0 % agarose gels in 0.5X TAE buffer, and the RNA was quantified with NanoDrop 2000 spectrophotometer (Thermo Scientific). One microgram of total RNA was treated with DNase I (Promega) according to manufacturer’s instructions and used for cDNA synthesis with oligo d(T) primer and the SuperScriptIII (Invitrogen) enzyme, according to manufacturer’s instructions. Quantitative PCR reactions were performed in a total volume of 20 μL, using 5 μL of a 20-fold diluted cDNA. The amplification reactions were performed using the SYBR® Select Master Mix (Applied Biosystems), according to manufacturer’s instructions. Primers used for GFP were qmGFP5_F3 (5’-AGTGGAGAGGGTGAAGGTGATGC-3’) and qmGFP5_R4 (5’- TCCCTCAGGCATGGCGCTCTT-3’). The genes Ubiquitin3 (Ubi3) and Elongation factor-1 α (EF-1) were used as reference genes, with primers previously described [39]. Three biological and technical replicates were used for all samples. Quantification of GFP expression levels was performed using the comparative CT method (ΔΔCT) through the Miner and qBase softwares [40‐42]. The t-student test was performed to compare the samples.
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Western blotting
Infiltrated Nicotiana benthamiana leaves were ground in liquid nitrogen and mixed with sample buffer (100 mM Tris [pH 6.8], 20 % glycerol, 4 % SDS, and 0.2 % bromophenol blue) containing 10 % β-mercaptoethanol [43]. Samples were then boiled at 90 °C for 10 min, and centrifuged for 5 min at 13,000 × g before loading on a gel. Extracts were run in 8 % SDS-PAGE gels for the detection of AtAGO1-Myc and in 12 % SDS-PAGE gels for detecting P19-myc, P0PL-AU-myc and P0CLs-myc with anti-myc antibody (1:2,000; Sigma, clone 9E10), followed by an anti-mouse HRP secondary antibody (1:5,000; Bio-Rad). Antibody–protein interactions were visualized using an enhanced chemiluminescence detection kit (GE Healthcare) according to the manufacturer’s instructions.
Acknowledgements
Authors acknowledge Dr. Jean-Lois Bélot and Dr. Rafael Galbieri, from Instituto Matogrossense do Algodão and Dr. Nelson Suassuna from EMBRAPA Algodão for sending the cotton infected plants. This study is part of the thesis research of RSC in pursuit of his PhD in Genetics at the Genetics Department of the Federal University of Rio de Janeiro. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) to GSM, MFSV and RLC. RSC and ILGA were supported by fellowships from CNPq.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
RSC, GSM, MFSV and RLC conceived and designed the experiments. RSC, ILGA and TFS performed the experiments. MFSV and TFS contributed with plant material. GSM, MFSV and RLC contributed reagents/materials/analysis tools. RSC and RLC wrote the manuscript. All authors read and approved the final manuscript.
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