Silenced Suppressor Genes Research Articles

Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus recently identified from the woody plant mulberry (Morus alba L.). Little is known about the functions of the proteins encoded by the MCLV genome. Here, all the MCLV-encoded proteins were examined for the ability to suppress gene silencing by an agroinfiltration assay in combination with northern blot analysis of green fluorescent protein (GFP) mRNA and western blot analysis. Of the six proteins, only one protein, V3, which has been predicted to play a role in viral movement, was found to suppress the gene silencing induced by a sense GFP gene in Nicotiana benthamiana 16c. The minimal amino acid sequence of V3 that maintains suppressor activity was also determined by constructing truncated mutants lacking different lengths of the amino acid sequences at the N- or C-terminus of the V3 protein. The results showed that the 94 N-terminal amino acid residues of V3 are sufficient to maintain V3 suppressor activity. In addition, the subcellular location of the V3 protein was investigated by confocal laser scanning microscopy after the expression of a V3-RFP fused protein in leaf epidermal cells of N. benthamiana. The results indicated that the V3 protein localized not only to the cytoplasm but also to the nucleus of N. benthamiana, implying that V3 can shuttle between the nucleus and the cytoplasm. Deletion mutant analysis indicated that a putative nuclear localization signal (NLS) between aa 118–134 might be responsible for the nuclear distribution of the V3 protein. Given the importance of RNA silencing in plant-virus interactions, the identification of a silencing suppressor of MCLV should be valuable in understanding the pathogenicity and molecular biology of this virus.

In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.

Functional Scanning of Apple Geminivirus Proteins as Symptom Determinants and Suppressors of Posttranscriptional Gene Silencing.

Apple geminivirus (AGV) is a recently identified geminivirus which is isolated from the apple tree in China. We carried out functional scanning of apple geminivirus proteins as symptom determinants and suppressors of posttranscriptional gene silencing (PTGS). Our results indicated that AGV V2 is an important virulence factor localized to the nucleus and cytoplasm that suppresses PTGS and induces severe symptoms of crinkling and necrosis. AGV C1 is also a virulence determinant which elicits systemic necrosis when expressed from a PVX-based vector. The AGV C4 is targeted to cytoplasm, plasma membrane, nucleus, and chloroplasts. The inoculation of PVX-C4 on N. benthamiana induced severe upward leaf curling, which implied that AGV C4 also functions as a symptom determinant, and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its subcellular localization and symptom development.

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.

Identification of an RNA silencing suppressor encoded by citrus vein enation virus

In order to know if any protein encoded by citrus vein enation virus (CVEV) might act as an RNA silencing suppressor (RSS), five open reading frames (ORF) of CVEV, including the coat protein (CP) gene, were individually cloned into the binary expression vector pBI121. The silencing suppression ability of the five ORFs was tested by ‘viral minireplicon infectivity assays’ based on a beet yellows virus (BYV) minireplicon carrying a reporter gene (BYVminiR-GFP). GFP imaging and microscopic analyses were used to observe whether RNA silencing suppression occurred or not. In addition, real-time quantitative RT-PCR was used to quantify the mRNA expression of GFP in the injected region. The fluorescence intensity of leaves with CP expression was stronger than that of the empty vector negative control, but it was weaker than that of the positive control HC-Pro of tobacco etch virus (TEV), indicating that the CP of CVEV partly inhibited the GFP gene silencing. Co-expression of each of the other four ORFs with BYVminiR-GFP showed an almost complete quenching of GFP fluorescence in infiltrated areas, similar to that of the negative control. The amount of GFP mRNA in the leaves co-expressed with CP was nearly twice as much as that of either the negative control or other ORFs. These results suggest that CP of CVEV is a weak gene silencing suppressor.

S-acylation mediates Mungbean yellow mosaic virus AC4 localization to the plasma membrane and in turns gene silencing suppression.

RNA silencing plays a critical role in plant resistance against viruses. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood. We examined the role of Mungbean yellow mosaic virus (MYMV) AC4 and showed that it is essential for infectivity but not for virus replication. It acts as a determinant of pathogenicity and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing whereas it does not interfere with local production of siRNA. We demonstrate the ability of AC4 to bind native 21–25 nt siRNAs in vitro by electrophoretic mobility shift assay. While most of the known VSRs have cytoplasmic localization, we observed that despite its hydrophilic nature and the absence of trans-membrane domain, MYMV AC4 specifically accumulates to the plasma membrane (PM). We show that AC4 binds to PM via S-palmitoylation, a process of post-translational modification regulating membrane–protein interactions, not known for plant viral protein before. When localized to the PM, AC4 strongly suppresses systemic silencing whereas its delocalization impairs VSR activity of the protein. We also show that AC4 interacts with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), a positive regulator of the cell-to-cell movement of RNAi. The absolute requirement of PM localization for direct silencing suppression activity of AC4 is novel and intriguing. We discuss a possible model of action: palmitoylated AC4 anchors to the PM by means of palmitate to acquire the optimal conformation to bind siRNAs, hinder their systemic movement and hence suppress the spread of the PTGS signal in the plant.

Multifunctional roles of geminivirus encoded replication initiator protein.

Geminivirus infection has been a threat to cultivation worldwide by causing huge losses to the crop. The single-stranded DNA genome of a geminivirus possesses a limited coding potential and many of the open reading frames (ORFs) are overlapping. Out of 5-7 ORFs that a geminivirus genome codes for, the AC1 ORF encodes for the replication initiator protein (Rep) which is involved in the replication of virus within the infected plant cell. Rep is the only viral protein absolutely required for the in planta viral replication. Across different genera of the Geminiviridae family, the AC1 ORF exhibits a high degree of sequence conservation thus it has been used as an effective target for developing broad spectrum resistance against the invading geminiviruses. This multifunctional protein is required for initiation, elongation as well as termination of the viral replication process. Rep is also involved in stimulation of viral transcription. In addition, it also functions as suppressor of gene silencing and is involved in the process of transcription by regulating the expression of certain viral genes. Rep protein also interacts with few viral proteins such as coat protein, replication enhancer protein and with several host factors involved in different pathways and processes for its replication and efficient infection. This review will summarise our current understanding about the role of this early viral protein in viral propagation as well as in establishment of pathogenesis in a permissive host.

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

TAS3 miR390-dependent loci in non-vascular land plants: towards a comprehensive reconstruction of the gene evolutionary history

Trans-acting small interfering RNAs (ta-siRNAs) are transcribed from protein non-coding genomic TAS loci and belong to a plant-specific class of endogenous small RNAs. These siRNAs have been found to regulate gene expression in most taxa including seed plants, gymnosperms, ferns and mosses. In this study, bioinformatic and experimental PCR-based approaches were used as tools to analyze TAS3 and TAS6 loci in transcriptomes and genomic DNAs from representatives of evolutionary distant non-vascular plant taxa such as Bryophyta, Marchantiophyta and Anthocerotophyta. We revealed previously undiscovered TAS3 loci in plant classes Sphagnopsida and Anthocerotopsida, as well as TAS6 loci in Bryophyta classes Tetraphidiopsida, Polytrichopsida, Andreaeopsida and Takakiopsida. These data further unveil the evolutionary pathway of the miR390-dependent TAS3 loci in land plants. We also identified charophyte alga sequences coding for SUPPRESSOR OF GENE SILENCING 3 (SGS3), which is required for generation of ta-siRNAs in plants, and hypothesized that the appearance of TAS3-related sequences could take place at a very early step in evolutionary transition from charophyte algae to an earliest common ancestor of land plants.

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana.

The exploration of emerging host organisms for the economic and efficient production of protein microbicides against HIV is urgently needed in resource-poor areas worldwide. In this study, the production of the novel HIV entry inhibitor candidate, griffithsin (GRFT), was investigated using Nicotiana benthamiana as the expression platform based on a non-viral vector. To increase the yield of recombinant GRFT, the RNA silencing defense mechanism of N. benthamiana was abolished by using three gene silencing suppressors. A transient expression system was used by transferring the GRFT gene, which encodes 122 amino acids, under the control of the enhanced CaMV 35S promoter. The presence of correctly assembled GRFT in transgenic leaves was confirmed using immunoglobulin-specific sandwich ELISA. The data demonstrated that the use of three gene silencing suppressors allowed the highest accumulation of GRFT, with a yield of 400 μg g-1 fresh weight, and this amount was reduced to 287 μg g-1 after purification, representing a recovery of 71.75%. The analysis also showed that the ability of GRFT expressed in N. benthamiana to bind to glycoprotein 120 is close to that of the GRFT protein purified from E. coli. Whole-cell assays using purified GRFT showed that our purified GRFT was potently active against HIV. This study provides the first high-level production of the HIV-1 entry inhibitor griffithsin with a non-viral expression system and illustrates the robustness of the co-agroinfiltration expression system improved through the use of three gene silencing suppressors.

AV2 protein of tomato leaf curl Palampur virus promotes systemic necrosis in Nicotiana benthamiana and interacts with host Catalase2

Tomato leaf curl Palampur virus (ToLCPalV) is a whitefly-transmitted, bipartite begomovirus. Here, we demonstrated that ectopic expression of AV2 from a Potato virus X (PVX)-based vector accelerated systemic necrosis and reactive oxygen species (ROS) accumulation in Nicotiana benthamiana. Furthermore, 10 amino acids from N-terminal region of AV2 were found to be associated with the systemic necrosis symptom/phenotype. Mutational studies of ToLCPalV infectious clones lacking the AV2 revealed that AV2 is essential for the systemic movement of DNA-A, symptom severity and viral DNA accumulation. In a yeast two-hybrid assay, Catalase2 (Cat2) was found to associate with AV2 protein. Further, silencing of Cat2 resulted in appearance of necrotic lesions on N. benthamiana and these plants were highly susceptible to ToLCPalV infection in comparison to control plants. Infection ToLCPalV on Solanum lycopersicum resulted in downregulation of Cat2 transcripts, followed by accumulation of ROS and stress marker transcripts. The AV2 protein also suppressed virus-induced gene silencing (VIGS) of the Phytoene desaturase (PDS) gene. Our results show that AV2 is essential for the pathogenicity, systemic movement and suppression of gene silencing in the host. Altogether, our findings suggest that interactions between AV2 and Cat2 might play a crucial role in the establishment of ToLCPalV infection.

Transient Transactivation Studies in Nicotiana benthamiana Leaves.

Transitory gene expression systems in Nicotiana benthamiana leaves, in combination with the use of gene silencing suppressors as the p19 or HC-pro proteins that allow for elevated levels of gene expression, have proven to be a highly versatile tool to analyze transcriptional function of DNA binding factors in the activated or repressed expression of their gene targets. This experimental setup uses Agrobacterium-mediated infection to deliver the various DNA constructs into the cell, and offers the advantage with respect to mesophyll protoplast transfection procedures that it entails a much easier protocol, in addition to preserving the intact leaf tissue, thus being more amenable to the study of wound and stress signaling pathways or to the functional analyses of regulators that respond to Ca+2 signatures. Furthermore, by using reporter constructs based on the LUCIFERASE (LUC) gene, which does not require a destructive determination assay, this expression system can be used to test for changes in gene activity over time or in response to various treatments, thus providing a comprehensive understanding of the signaling pathways that modulate activity of the expressed regulators and therefore their in vivo function in the control of the analyzed promoter.

Identification of host cellular targets of AC4 and AV2 proteins of tomato leaf curl palampur virus and their sub-cellular localization studies.

Tomato leaf curl palampur virus (ToLCPalV) is a bipartite begomovirus with genome organization typical of old world begomoviruses. It infects commercially important crops and weeds in the Asian subcontinent. Apart from other proteins, the DNA-A of the virus encodes AV2 and AC4 proteins of approximately 13.73 and 6.7kDa, respectively. In case of other begomoviruses, previous studies have shown the role of AV2 and AC4 proteins in virus movement, pathogenesis and suppression of gene silencing. However, the ToLCPalV proteins are significantly variable in comparison to closest relative and hence there is a need to work out their functions. In this study, we identified 9 cellular proteins of tomato that interact with AV2 and AC4 proteins, through yeast two hybrid screening. Upon sequence analysis, these interactors were identified as cysteine protease, katanin p60 ATPase-containing subunit A-like, guanine deaminase, NADH dehydrogenase (ubiquinone) iron-sulfur protein, glyceraldehyde-3-phosphate dehydrogenase B, 60S acidic ribosomal P0 protein, acyl co-A dehydrogenase IBR3, oxygen-evolving enhancer protein 1 and peroxisomal membrane protein 11D. These proteins play a vital role in protein degradation, plant defense response, microtubule severing, photosynthesis and protein synthesis. The two viral proteins, however, did not interact with each other in yeast. AV2 when fused with GFP under the control of cauliflower mosaic virus 35S promoter was localized in nucleus and cytoplasm. On the other hand, AC4-GFP fusion was localized only in cytoplasm. The outcome of present study will help to elucidate the mechanism of viral pathogenesis. Further functional characterization of identified host proteins will provide an insight into their involvement in disease development.

Potyviral Gene-Silencing Suppressor HCPro Interacts with Salicylic Acid (SA)-Binding Protein 3 to Weaken SA-Mediated Defense Responses.

The viral infection process is a battle between host defense response and pathogen antagonizing action. Several studies have established a tight link between the viral RNA silencing suppressor (RSS) and the repression of salicylic acid (SA)-mediated defense responses, nonetheless host factors directly linking an RSS and the SA pathway remains unidentified. From yeast two-hybrid analysis, we identified an interaction between the potyviral RSS helper-component proteinase (HCPro) and SA-binding protein SABP3. Co-localization and bimolecular fluorescence complementation analyses validated the direct in vivo interaction between Turnip mosaic virus (TuMV) HCPro and the Arabidopsis homologue of SABP3, AtCA1. Additionally, transient expression of TuMV HCPro demonstrated its ability to act as a negative regulator of AtCA1. When the plants of the AtCA1 knockout mutant line were inoculated with TuMV, our results indicated that AtCA1 is essential to restrict viral spreading and accumulation, induce SA accumulation, and trigger the SA pathway. Unexpectedly, the AtCA1 overexpression line also displayed a similar phenotype, suggesting that the constitutive expression of AtCA1 antagonizes the SA pathway. Taken together, our results depict AtCA1 as an essential regulator of SA defense responses. Moreover, the interaction of potyviral HCPro with this regulator compromises the SA pathway to weaken host defense responses and facilitate viral infection.

Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein

The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

An Insight into Cotton Leaf Curl Multan Betasatellite, the Most Important Component of Cotton Leaf Curl Disease Complex.

Cotton leaf curl disease (CLCuD) is one of the most economically important diseases and is a constraint to cotton production in major producers, Pakistan and India. CLCuD is caused by monopartite plant viruses belonging to the family Geminiviridae (genus Begomovirus), in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMuB) belonging to a newly-established family Tolecusatellitidae (genus Betasatellite). CLCuMuB has a small genome (ca. 1350 nt) with a satellite conserved region, an adenine-rich region and a single gene that encodes for a multifunctional βC1 protein. CLCuMuB βC1 protein has a major role in pathogenicity and symptom determination, and alters several host cellular functions like autophagy, ubiquitination, and suppression of gene silencing, to assist CLCuD infectivity. Efficient trans-replication ability of CLCuMuB with several monopartite and bipartite begomoviruses, is also associated with the rapid evolution and spread of CLCuMuB. In this article we comprehensively reviewed the role of CLCuMuB in CLCuD, focusing on the βC1 functions and its interactions with host proteins.

V2 from a curtovirus is a suppressor of post-transcriptional gene silencing.

The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway.

Tomato Leaf Curl New Delhi Virus: An Emerging Virus Complex Threatening Vegetable and Fiber Crops.

The tomato leaf curl New Delhi virus (ToLCNDV) (genus Begomovirus, family Geminiviridae) represents an important constraint to tomato production, as it causes the most predominant and economically important disease affecting tomato in the Indian sub-continent. However, in recent years, ToLCNDV has been fast extending its host range and spreading to new geographical regions, including the Middle East and the western Mediterranean Basin. Extensive research on the genome structure, protein functions, molecular biology, and plant–virus interactions of ToLCNDV has been conducted in the last decade. Special emphasis has been given to gene silencing suppression ability in order to counteract host plant defense responses. The importance of the interaction with DNA alphasatellites and betasatellites in the biology of the virus has been demonstrated. ToLCNDV genetic variability has been analyzed, providing new insights into the taxonomy, host adaptation, and evolution of this virus. Recombination and pseudorecombination have been shown as motors of diversification and adaptive evolution. Important progress has also been made in control strategies to reduce disease damage. This review highlights these various achievements in the context of the previous knowledge of begomoviruses and their interactions with plants.

SGS3 Cooperates with RDR6 in Triggering Geminivirus-Induced Gene Silencing and in Suppressing Geminivirus Infection in Nicotiana Benthamiana.

RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs) mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3), was also found to fight against some viruses in Arabidopsis. In this study, we showed that SGS3 from Nicotiana benthamiana (NbSGS3) is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS) and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS) and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6) silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana, which provides valuable information for breeding geminivirus-resistant plants.

The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity.

Cucumber mosaic virus (CMV) is a model virus for plant–virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.