Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates
© Cascardo et al. 2015
Received: 28 April 2015
Accepted: 4 August 2015
Published: 12 August 2015
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).
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.
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.
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.
Cotton leafroll dwarf virus (CLRDV) is the causal agent of an economically important cotton (Gossypium hirsutum) disease called cotton blue disease (CBD) . Aphis gossypii-transmitted CBD has been observed in several cotton-producing areas of Central Africa, Asia and South America . 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 . 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) . 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 . 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 .
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 . 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 . 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 . 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 . 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 . 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 . 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 .
Recently, the P0 protein from an Argentinian isolate of CLRDV (P0CL-ARG) has been characterized as a RNA silencing suppression protein . 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 . 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
Brazilian isolates of cotton leafroll dwarf virus used in the study
G. hirsutum cultivar
CBD resistance phenotype
Primavera do Leste – MT
Acreuna – GO
Holambra – SP
Campo Verde - MT
Ipameri - GO
Palotina - PR
Patos de Minas - MG
Percentage of amino acid identity among the viral isolates used in the study
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 . 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) . 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 , leading to a 35S-driven expression of Myc-tagged 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) . Therefore, the local silencing suppression variability observed among CLRDV P0s is probably associated with alternative functional residues.
Suppression of systemic silencing by CLRDV P0s
Proportion of plants showing systemic silencing at 16, 20 and 29 days post-infiltration (dpi)
GFP + Vector
GFP + P0PL-AU
GFP + P0CL-PV1
GFP + P0CL-ARG
GFP + P0CL-Acr9
GFP + P0CL-Hol1
GFP + P0CL-Ima2
GFP + P0CL-Ipa4
GFP + P0CL-Pal3
GFP + P0CL-Pm1
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  and for the P0s from sugarcane yellow leaf virus and beet western yellows virus .
AGO destabilization by CLRDV P0s
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.
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 . 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  and the P19 coding sequence  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 .
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 , resulting in 35S-driven, Myc-tagged proteins when expressed in plants.
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 . Trees were constructed by the neighbor-joining (NJ) method , with the pair-wise deletion option and number of differences matrix.
Agrobacterium tumefaciens, strain GV3101, were infiltrated in Nicotiana benthamiana leaves as described previously . 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)  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 , 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.
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 . 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.
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 . 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.
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.
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