ABSTRACT
Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses.
IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward first understanding how the viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses, including Bamboo mosaic virus, Foxtail mosaic virus, and Potato virus X. Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.
INTRODUCTION
Bamboo mosaic virus (BaMV) is a positive-strand monopartite RNA virus belonging to the Potexvirus genus. The genome of BaMV contains approximately 6,400 nucleotides on which five open reading frames (ORFs) are organized, plus a 5′ cap 0 structure and a 3′ poly(A) tail (1). ORF1 of BaMV encodes a nonstructural protein of 1,366 amino acids, termed REPBaMV, which is essential for replication/transcription of BaMV. Catalytic properties of the functional domains constituting REPBaMV have been intensively studied, and the results were summarized in a recent review (2). In brief, the domain at the N-terminal one-third is an mRNA capping enzyme exhibiting a unique AdoMet-dependent guanylyltransferase activity. The central helicase-like domain possesses an activity of cleaving the 5′-γ-phosphate off the nascent viral RNA transcripts, through which the RNA transcripts are ready to accept m7GMP from the m7GMP-conjugated capping domain to complete the formation of 5′ cap structure. The C-terminal domain is an RNA-dependent RNA polymerase (RdRp) domain with preference for positive- and negative-stranded RNAs of BaMV. ORF2, -3, and -4 are overlapped, and their translational products are essential for the spread of BaMV within host plants (3). ORF5 encodes the 25.4-kDa coat protein (CP), which also plays a critical role in the viral cell-to-cell movement via an interaction with the helicase-like domain of REPBaMV (4). A couple of subgenomic RNAs (sgRNA), coterminal with the viral 3′-untranslated region, are produced upon BaMV infection in the host cells (5). The ∼2-kb sgRNA primarily directs the synthesis of the product of ORF2, a 27.6-kDa movement protein, whereas the abundant ∼1-kb sgRNA is for the production of CP.
In nature, a clade of BaMV-associated RNA molecules of ∼836 nucleotides is found repeatedly (6). Those satellite RNAs, termed satBaMV, contains only one ORF that encodes a 20-kDa polypeptide (P20). P20 is dispensable for satBaMV replication; however, it does play an important role for satBaMV to accumulate in systemic leaves (7). Enzymatic probing suggested that the secondary structures of the 3′-untranslated region of satBaMV and BaMV are similar (8). Cotranscription of the satBaMV SF4 variant and a binary plasmid-based REPBaMV expression cassette via agroinfiltration in Nicotiana benthamiana leaves elevates the expression of REPBaMV from barely discernible to notable levels in Western blotting assays (9). Presumably, SF4 acts as a RNA scaffold on which REPBaMV can fold correctly and/or be sequestered from protease degradation. Moreover, SF4 may facilitate the recruitment of host factors to constitute a competent viral replication complex. In fact, the REPBaMV-containing membrane fraction prepared from the agroinfiltrated N. benthamiana has facilitated the in vitro RdRp activity assay by using the endogenous SF4 as the template (9, 10). It is worth noting that addition of ionic detergents, e.g., 0.1% sodium dodecyl sulfate (SDS), in the REPBaMV-containing membrane preparation was able to boost the in vitro BaMV RdRp activity, suggesting a strong physical stability of the membrane-associated viral replication complex. This notion is consistent with the general observation that replication complexes of most plant viruses are embedded in membrane-enclosed microcompartments (11–13).
Limited by the small genome size, a plant RNA virus needs a dynamic array of host factors to fulfill the various steps of its multiplication cycle (13–15). Great progress in identification of host factors and the understanding of their assisting functions during various stages of viral infection have been made over the past few decades. Specifically, these discoveries focused on the following: viral particle disassembly, membrane remodeling, and formation of the viral replication complex, viral genome translation and replication, viral cell-to-cell movement and long-distance transport. Understanding these mechanisms of viral infection could not have been compiled without studying viruses such as Brome mosaic virus (BMV) (14), Tobacco mosaic virus (TMV) (13), Tomato bushy stunt virus (TBSV) (12), and Red clover necrotic mosaic virus (RCNMV) (16). Consequently, these above-mentioned viruses have become valuable references for many other plant RNA viruses. On the other hand, a variety of host proteins can be recruited via recognition by the viral proteins or nucleic acids to execute an otherwise novel antiviral activity. Therefore, finding cellular proteins that constitute the viral replication complex or interact directly with the viral replication proteins would pave the way toward understanding how the viral replication is modulated. Regarding the interplay between BaMV and N. benthamiana, the host factors known to regulate the viral RNA replication and movement are discussed in a recent review (17). The factors specifically affecting REPBaMV are briefly described herein. A putative methyltransferase, which negatively modulates BaMV accumulation, was identified through yeast two-hybrid (Y2H) screening by using the RdRp domain of REPBaMV as the bait (18). Y2H also showed an interaction between REPBaMV and heat shock protein 90, through which the replication efficiency of BaMV could be increased (19). An immunoprecipitation approach using REPBaMV-specific antiserum also helped identify a number of potential host factors from the Sarkosyl-solubilized REPBaMV-containing membrane fraction. Among them, a cytoplasmic 5′→3′ exoribonuclease is capable of increasing BaMV accumulation by presumably removing uncapped aberrant viral RNAs, which otherwise might interfere with the replication of normal viral RNAs (9). The N. benthamiana mitogen-activated protein kinase phosphatase 1 (NbMKP1), a negative regulator in the plant mitogen-activated protein kinase (MAPK) pathway, attenuates the replication efficiency of BaMV, suggesting that an environment favorable for BaMV replication is created by the activation of MAPK pathway (10).
Prompted by the resistance of the in vitro SF4-dependent RNA polymerase activity to 0.1% SDS, a REPBaMV-associated protein complex was isolated from a SDS-polyacrylamide gel electrophoresis (PAGE) gel, and the proteins within were identified using tandem mass spectrometry (MS/MS) in this study. The relevance of the targeted proteins to BaMV replication were subsequently screened by inoculating a recombinant BaMV virion that carries the green fluorescent protein (GFP) gene on leaves of the targeted gene-silenced N. benthamiana. Those with apparent effects on BaMV replication-dependent GFP expression were then chosen for further studies. Among the screened proteins, proliferating cell nuclear antigen (PCNA) was found to have a profound effect on BaMV replication. PCNA is essential for biological processes such as DNA replication, DNA repair, and cell cycle control (20–22). In DNA replication, PCNA encircles double-stranded DNA by means of its trimeric ring-shaped structure and interacts with DNA polymerase δ or ε to ensure the processivity of DNA polymerase (23, 24). Despite being originally identified as a nuclear antigen, PCNA may carry out other noncanonical jobs in the cytoplasm. For instance, a substantial amount of PCNA is also present in the cytoplasm of cancer cells, where it interacts with several cytoplasmic oncoproteins and some glycolytic enzymes (25). Interestingly, Arabidopsis thaliana PCNA was found in both the nucleus and the cytoplasm according to a bimolecular fluorescence complementation assay (26). Hence, the effect of plant PCNA on BaMV replication in protoplasts was thoroughly investigated, and the underlying mechanism is addressed in this report.
RESULTS
Host proteins affiliated with the REPBaMV-associated protein complex.Proteins in the P30 fraction prepared from leaves agroinfiltrated with pERep-HA and pKSF4 were separated by 4 to 13% gradient SDS-PAGE as described in Materials and Methods. REPBaMV-HA appeared at two migration positions as indicated by Western blotting using anti-HA antibody (Fig. 1A). Apparently, two close antibody-recognized bands were at the position slightly above 150 kDa, consistent with the molecular mass of hemagglutinin-expressing REPBaMV (REPBaMV-HA). One more banding appeared at the very upper position of the polyacrylamide gel. This upper band might reflect the residual BaMV replication complex, rather than protein precipitates, under the denaturing electrophoretic condition, because it was actually within the separation gel. This presumption is supported by our observation that the in vitro RdRp activity of REPBaMV, exhibited by the P30 fraction prepared from the BaMV-infected N. benthamiana leaves, had a much stronger reaction in the buffer that contained 0.3% Sarkosyl or 0.1% SDS (Fig. 1B). Thus, the gel at the very upper position was sliced and the proteins within were identified by tandem mass spectrometry. The corresponding position on the same gel of the control sample, which was from leaves agroinfiltrated with only pKSF4, was also sliced and subjected to mass spectrometric analysis. After subtractive comparison, host proteins that showed up only in the tested sample, but not in the control, were listed (Table 1). We assumed that some of the proteins might take part in modulating the viral replication activity, while the others were bystanders merely because they were simultaneously present in the sliced gel. Hence, functional screening of the candidates for their relevance to BaMV replication was necessary.
Protein profiles of P30 analyzed by SDS-PAGE. (A) The leaf membrane fraction P30 of N. benthamiana was electrophoresed using SDS-PAGE (4 to 13% gradient polyacrylamide), followed by silver staining and Western blot analysis using antihemagglutinin antibody (Sigma-Aldrich). Samples 1 and 2 denote Nicotiana leaves agroinfiltrated with pKSF4 and pKSF4 plus pERep-HA, respectively. The arrow points to the putative replication complex of REPBaMV shown in the Western blot, and the rectangle with a dashed border indicates the place at which the gel was sliced for protein identification by tandem mass spectrometry. (B) Detergent dependence of the RdRp activity exhibited by REPBaMV. P30 extracted from BaMV-infected N. benthamiana was suspended in TG buffer containing the indicated detergents. The reaction conditions and RNA product analysis were described in Materials and Methods. gRNA and sgRNA denote genomic and subgenomic RNA of BaMV, respectively.
Silencing effects of potential host factors of BaMV on N. benthamiana development and BaMV infectivity
Virus-induced gene silencing (VIGS) was performed in N. benthamiana using Tobacco rattle virus vectors to knock down the expression of each of the candidate genes. VIGS experiments showed that several candidates listed in Table 1 are of importance for the growth and leaf development of N. benthamiana. Silencing of those proteins, including eukaryotic initiation factor 4A-14, glycolate oxidase, chloroplast zebra-necrosis protein, RabE1, MAPKKKε, nitrate reductase, the neuroblastoma-amplified gene protein, and plastid RNA-binding protein caused growth retardation and/or changes in leaf morphology (Table 1). Considering the abnormality in leaf development and the difficulty in virus inoculation, those gene-silenced plants were therefore devoid of BaMV challenge. On the other hand, the plants without notorious changes in phenotype after silencing treatments were then inoculated with the recombinant BaMV (BaMV-GFP) derived from the infectious clone pCBG. The numbers and sizes of the green fluorescent foci on the inoculated leaves were then used as indexes to screen the candidates that are more likely involved in the viral replication. In this primary screen, PCNA was particularly noted because bare GFP foci appeared in the inoculated leaves (see the following text), and this observation prompted us to further investigate the probable role of PCNA in BaMV replication.
Presence of PCNA in the BaMV replication complex.Considering most of the documented functions of PCNA occur in the nucleus, it was important to demonstrate a plausible interaction between PCNA and the BaMV replication complex in the cytoplasm. That accumulation of REPBaMV-HA in N. benthamiana was greatly enhanced by simultaneous expression of satBaMV SF4 was restated in this study (Fig. 2A), although it was reported before (9). To prove the in vivo interaction between PCNA and REPBaMV-HA in the BaMV replication complex, binary plasmids pERep-HA, pKSF4, and pE-PCNA were cointroduced into N. benthamiana via agroinfiltration. pE-PCNA was replaced with pE-OFP, which encodes orange fluorescent protein (OFP), in the control. The P30 fraction isolated from the agroinfiltrated leaves was solubilized in buffer containing 0.3% Sarkosyl, and the accumulation of REPBaMV-HA and PCNA in P30 was analyzed by Western blotting using specific anti-HA and anti-PCNA antibodies (Fig. 2B and C, respectively). The expression of REPBaMV-HA and PCNA was promoted as expected. A relatively small amount of PCNA was detected also in the control leaf, which could be attributed to the chromosomal PCNA genes (Fig. 2C, lane 1). The supernatant of the Sarkosyl-solubilized P30 was incubated with specified antibody, followed by addition of protein G-conjugated magnetic Sepharose beads. After thorough washing, the collected beads were then assayed for the presence of PCNA or REPBaMV-HA by Western blotting. PCNA was clearly detected in the anti-HA antibody-precipitated sample prepared initially from the leaves agroinfiltrated with pERep-HA, pKSF4, and pE-PCNA (Fig. 2D). No PCNA was detected when anti-His antibody was used in the immunoprecipitation step. Similarly, REPBaMV-HA was detected in the bead-collected sample only when anti-PCNA antibody, but not anti-His antibody, was used in the immunoprecipitation step (Fig. 2E). The positive results of the coimmunoprecipitation experiments support the notion that some PCNA molecules were able to interact with the BaMV replication complex in the cytoplasm.
Interaction of PCNA with REPBaMV-HA in P30 extracted from N. benthamiana. Plant leaves were agroinfiltrated with pERep-HA and pKSF4 to express REPBaMV-HA and satBaMV SF4, respectively. Meanwhile, PCNA or OFP was additionally expressed by agroinfiltration with pE-PCNA or pE-OFP, respectively, when it was necessary. The P30 fraction extracted from the leaves 2 days post-agroinfiltration was analyzed by Western blotting using anti-PCNA antibody or anti-HA antibody. (A) Dependence of REPBaMV-HA accumulation on the presence of satBaMV SF4. Molecular masses of the protein standards are indicated along the left side. (B) Detection of REPBaMV-HA in P30 fractions by using anti-HA tag antibody. (C) Detection of PCNA in P30 fractions by using anti-PCNA antibody. rPCNA was the purified PCNA produced by the recombinant E. coli strain. (D) The 0.3% Sarkosyl-solubilized supernatant of P30 was precipitated by anti-HA or anti-His antibodies, followed by Western blotting using anti-PCNA antibody. (E) The supernatant was precipitated by anti-PCNA or anti-His antibodies, followed by Western blotting using anti-HA antibody. The arrow in panel D points to PCNA coprecipitated with REPBaMV-HA, while the arrow in panel E points to REPBaMV-HA coprecipitated with PCNA.
Effects of PCNA silencing on the development of plant leaves.PCNA silencing affected N. benthamiana in two aspects: first, the plants had shorter stem internodes, and second the leaves became thicker and turned light green at areas distant from veins (Fig. 3A). Notwithstanding the phenotypic differences, the PCNA-silenced plants were still challenged with BaMV virions. Much less green fluorescent foci appeared on the PCNA-silenced leaves in comparison with the control (Fig. 3B), suggesting that either the leaf structure was more resistant to virus inoculation or a hostile cellular condition was established against virus replication and/or cell-to-cell spreading. The decrease in the PCNA transcript in PCNA-silenced plants was confirmed by real-time quantitative PCR (qPCR) (Fig. 3C). To learn more about the changes, the leaves were observed under transmission electron microscope. Interestingly, chloroplasts within the PCNA-silenced cells were packed with large starch granules (Fig. 4A). Iodine stain confirmed the extraordinary accumulation of starch in the PCNA-silenced leaves (data not shown). Next, we tried to prepare protoplasts from the PCNA-silenced leaves so that the replication efficiency of BaMV could be examined at the single-cell level. Surprisingly, a large fraction of protoplasts prepared from the PCNA-silenced leaves were devoid of intact chloroplasts and accompanied by many free chloroplasts without chlorophyll in the preparation (Fig. 4B). Disruption of the chloroplast membrane by the aforementioned large starch granules under the high centrifugal force of protoplast preparation might have accounted for this abnormal outcome. Considering the global impact caused by PCNA silencing, it was improbable to ascribe the limited infectivity of BaMV, as shown in Fig. 3B, directly to PCNA itself. In other words, this limitation might represent an indirect result due to combined unfavorable factors such as the fragile nature of chloroplast and global composition changes in the PCNA-silenced leaves.
Effects of PCNA silencing on the development of N. benthamiana and BaMV infectivity. Downregulation of the target gene was achieved through Tobacco rattle virus-induced gene silencing as described in Materials and Methods. (A) Phenotypes of the PCNA-silenced plant and the mock control plant. (B) Appearance of green fluorescent foci on leaves of the silenced plants 4 days after inoculation with BaMV-GFP. (C) The relative transcription levels of PCNA, assayed by real-time qPCR, in PCNA-silenced and the control plants.
Morphological changes in chloroplasts and protoplasts upon PCNA silencing. (A) Leaves from the indicated gene-silenced N. benthamiana were collected and observed under transmission electron microscope. (B) Protoplasts were prepared from the indicated gene-silenced leaves and observed under microscope (bright field).
Inhibition of BaMV replication by PCNA in healthy protoplasts.An alternative approach was taken by examining the influence of PCNA on BaMV replication in protoplasts prepared from healthy N. benthamiana plants. The BaMV infectious clone pCBG was used to initiate the BaMV replication cycle, and the PCNA expression plasmid pBI-PCNA was introduced to additionally produce PCNA in protoplasts. Plasmid pBI221, which constitutively produces β-glucuronidase, was used as the control to substitute for pBI-PCNA. Protoplasts were transfected with combinations of pCBG, pBI-PCNA, and pBI221 as indicated, and subsequently the accumulation of BaMV CP in protoplasts 20 h posttransfection was analyzed by Western blotting (Fig. 5A). The effect of PCNA was remarkable: the more pBI-PCNA was used in transfection, the less CP was accumulated in protoplasts. This result suggests that PCNA had a high propensity to inhibit BaMV replication. To ensure that pBI-PCNA truly produced additional PCNA in protoplasts, pBI-PCNA-H6, designed to produce C-terminally 6×His tag-fused PCNA, was also used in the protoplast experiment. Transfection with pBI-PCNA-H6 also resulted in remarkable inhibition of the accumulation of BaMV CP (Fig. 5B), while expression of the recombinant PCNA was confirmed by Western blotting using anti-hexahistidine tag antibody (Fig. 5B). As with many plant RNA viruses, BaMV replicates in the cytoplasm. To work as an infectious clone, the recombinant BaMV cDNA carried in pCBG must be transcribed first by plant RNA polymerase in the nucleus to produce the positive transcript of the virus, which was then exported into cytoplasm to set up the viral replication process. Since PCNA is well known for its essential role in DNA replication, we wondered whether PCNA unexpectedly interfered with the production and/or export of BaMV transcript, consequently stopping the replication process of BaMV. To test this possibility, BaMV genomic RNA (gRNA) was used as a substitute for pCBG in the protoplast transfection experiment. The remarkable inhibition effect of PCNA on the accumulation of BaMV CP was reproducible (Fig. 5C), indicating that PCNA does have a strong inhibitory effect on BaMV replication.
Decrease of BaMV CP upon additional expression of PCNA in protoplasts. Protoplasts (1 × 105) derived from healthy N. benthamiana leaves were transfected with the indicated plasmids, and accumulation of BaMV CP was assayed by Western blotting 20 h after transfection. (A) Protoplasts were cotransfected with different combinations of pCBG, pBI-PCNA, and pBI221. Each experimental condition had three biological replicate samples. (B) Protoplasts were cotransfected with 1 μg pCBG and 3 μg pBI-PCNA-H6 or pBI221 as indicated. Each experimental condition had three biological replicate samples. The expression of C-terminally 6×His tag-fused PCNA (PCNA-H6) in the transfected protoplasts was evidenced by Western blotting using anti-6×His tag antibody (GeneTex). (C) Protoplasts were cotransfected with 0.5 μg BaMV gRNA and 3 μg pBI-PCNA or pBI221. Each experimental condition contained four biological replicate samples.
Foxtail mosaic virus (FoMV), phylogenetically close to BaMV, shares several similar host-pathogen interactions to BaMV when they proliferate in N. benthamiana. For example, the plant MAPK phosphatase 1, a negative regulator in the MAPK pathway, reduces the replication efficiency of both BaMV and FoMV (10), and the cytoplasmic 5′→3′ exonuclease increases the accumulation level of BaMV as well as FoMV (9). The effect of PCNA on FoMV was also tested in this study. Similar to the case of BaMV (Fig. 6A), PCNA also reduced the accumulation level of FoMV CP, although to a lesser extent (Fig. 6B). In another experiment, the inhibitory effect of PCNA on PVX replication in protoplasts was examined. The accumulation of PVX CP in protoplasts was significantly decreased when the viral infectious clone pXTAL was cotransfected with pBI-PCNA, rather than pBI221 (Fig. 6C). These results imply that the inhibitory function of PCNA may apply to many other potexviruses.
Decrease of BaMV CP, FoMV CP, as well as PVX CP upon additional expression of PCNA in protoplasts. Protoplasts (1 × 105) derived from healthy N. benthamiana leaves were cotransfected with 1 μg viral infectious clone and 3 μg pBI-PCNA or pBI221. The accumulation level of the specified viral CP was analyzed by Western blotting 20 h after transfection. pCBG, pCF, and pXTAL were used as infectious clones to determine the effect of PCNA on BaMV (A), FoMV (B), and PVX (C), respectively.
Mutational effects of PCNA on BaMV replication.PCNA, also known as DNA clamp, acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. In regards to DNA replication, PCNA assists DNA polymerase δ and ε to continuously hold DNA during DNA replication by interacting with replication factor C (RFC). Thus, the presence of PCNA is essential for DNA synthesis and maturation of Okazaki fragments. To determine whether the DNA replication-assisting function of PCNA was required for the inhibitory function of BaMV replication, couple critical residues of PCNA were substituted with alanine in this study. Specifically, Asp41 which is a critical residue for PCNA to stimulate DNA polymerase δ and RFC ATPase activity (27), and Phe114 (corresponding to Tyr114 in human PCNA) a critical residue to stabilize PCNA in the homotrimeric form (28). The effects of the mutant PCNAs on the replication efficiency of BaMV were investigated using Northern blotting assays (Fig. 7). Similar to the effect on BaMV CP, the accumulations of both the genomic and subgenomic RNAs of BaMV were strongly suppressed by PCNA. However, the mutant PCNAs, both D41A and F114A, were deprived of their activity against the accumulation of BaMV RNAs, suggesting that the inhibitory function of PCNA against BaMV replication pertains to its ring-shaped trimeric structure.
Debilitation of PCNA’s function in BaMV replication by D41A and F114A mutations. Protoplasts (1 × 105) derived from healthy N. benthamiana leaves were cotransfected with 1 μg pCBG and 3 μg pBI-PCNA, pBI-PCNA(D41A), pBI-PCNA(F114A), or pBI221. Each experimental condition had three biological replicate samples. The accumulation of BaMV gRNA and sgRNAs in protoplasts 20 h after transfection was analyzed by Northern blotting using probe specifically recognizing the coterminal 3′-UTR.
Negative correlation between PCNA and BaMV replication in plant.PCNA is actively expressed in the growing point of plants (22). Whether a negative correlation exists between PCNA expression and BaMV replication in N. benthamiana was an interesting question. A transgenic line of N. benthamiana carrying a transgene composed of the entire cDNA of BaMV-GFP and a preceding 35S promoter was used to find the answer. All the leaves of the transgenic plant were taken for analysis of PCNA and BaMV-encoded proteins such as GFP and CP (Fig. 8). Supposedly, the recombinant BaMV transcript could be produced throughout the transgenic plant; however, GFP was found mainly in older leaves (Fig. 8A, numbering 1 to 4). GFP was barely noticed in relatively younger leaves (numbering 6 to 8) where PCNA was strongly expressed, as indicated by Western blotting using anti-PCNA antibody (Fig. 8B). CP had a similar expression pattern to GFP. Since this analysis did not involve virus inoculation, the inability to generate GFP foci and to accumulate CP in young leaves should be due to an unfavorable cellular environment for BaMV replication rather than a difficulty in cell-to-cell spreading. Taken together with the data obtained from the protoplast experiments, PCNA seems to be a critical factor rendering the cells in younger leaves hostile to BaMV replication.
Negative correlation in protein expression profiles between PCNA and BaMV-encoded proteins. (A) GFP expression in transgenic N. benthamiana that carries the cDNA of BaMV-GFP. Green fluorescence emitting from leaves of 40-day-old plants was recorded using the Fujifilm luminescent image analyzer LAS-4000. The leaves were numbered from the oldest to the newest in the order of 1 to 8. (B) Total proteins of leaves were analyzed by 10% SDS-PAGE. Gels were stained with Coomassie blue to show the amounts of BaMV CP (CBS) or transferred to polyvinylidene difluoride (PVDF) membrane and immunoblotted with the PCNA-specific antibody to indicate the expression of PCNA (WB). NT denotes the sample derived from leaf 5 of a nontransgenic N. benthamiana plant.
Inhibition of REPBaMV activity by PCNA.To determine the mechanism by which BaMV replication was inhibited, the effect of PCNA on the in vitro REPBaMV activity was investigated. The recombinant PCNA-H6 of N. benthamiana was expressed in Escherichia coli and purified by immobilized metal affinity and gel filtration chromatographies (Fig. 9A). Meanwhile, the P30 fraction was isolated from N. benthamiana leaves that had been agroinfiltrated with binary plasmids pERep-HA and pKSF4. The SF4-dependent RNA polymerase activity exhibited by REPBaMV in P30 was assayed under the influence of the E. coli-expressed PCNA-H6. Inclusion of the wild-type PCNA-H6 in the reaction mixture significantly decreased the production of radiolabeled RNA product of SF4 in comparison with the effect of bovine serum albumin (Fig. 9B). Moreover, the more PCNA-H6 was included in the reaction mixture, the stronger inhibitory effect on polymerase activity was observed. It is noteworthy that the purified PCNA preparation was not contaminated with E. coli nucleases (data not shown); therefore, the observed inhibition effect of PCNA was authentic. The mutant PCNA-H6, either D41A or F114A, was devoid of the inhibitory effect on the polymerase activity of REPBaMV, suggesting that the two different tasks performed by PCNA (i.e., assisting DNA replication and suppressing BaMV replication) require a common structure of PCNA.
Suppression of the in vitro BaMV RdRp activity by PCNA. (A) Purification of the E. coli-expressed C-terminally hexahistidine tag-fused PCNA (PCNA-H6). The recombinant PCNA was purified from cell extracts through steps of immobilized metal affinity chromatography (IMAC) and gel filtration chromatography. (Left panel) Proteins in the cell extract of E. coli harboring pET29-PCNA and the elution solution from IMAC were analyzed by SDS-PAGE. (Right panel) The purified PCNAs, including the wild type and D41A and F114A mutants, were analyzed by SDS-PAGE. (B) The P30 membrane fraction was isolated from N. benthamiana leaves that had been agroinfiltrated with pERep-HA and pKSF4, and its SF4 RNA-dependent RNA polymerase activity was measured in the presence or absence of PCNA as indicated. The 32P-labeled RNA products were electrophoresed on an agarose gel.
PCNA encircles DNA in its trimeric form when it participates in DNA replication and DNA repair. Therefore, it is logical to speculate that PCNA has an ability to bind the viral RNA; as a result, it interferes with the correct assembly of the viral replication complex or blocks the replication process. To test this possibility, an aliquot of oligo(dT)-conjugated magnetic beads was premixed with BaMV gRNA, and then the purified PCNA-H6 was included in the mixture for further incubation. After repeatedly washing with buffer, the amount of PCNA-H6 that was still associated with the beads was analyzed by SDS-PAGE. A substantial fraction of PCNA-H6 could be recovered from the beads, and more PCNA-H6 was obtained when more BaMV gRNA was included in the mixture (Fig. 10A). No PCNA-H6 was recovered from the beads if the viral RNA was absent in the mixture, excluding the possibility of a direct interaction between PCNA and the beads. Taken together, PCNA-H6 has a capacity to bind BaMV gRNA, which in turn associates with the beads through hybridization between its 3′ poly(A) tail and the bead-coupled oligo(dT). It is noteworthy that the viral RNA-binding ability of PCNA was abolished by the mutations of D41A and F114A (Fig. 10B), suggesting that the viral RNA-binding capacity is prerequisite for PCNA to suppress BaMV replication.
BaMV gRNA-mediated pulldown of PCNA by using oligo(dT)-conjugated beads. (A) BaMV gRNA at the indicated amounts (0, 1, or 3 μg) was mixed with 200 μl oligo(dT)15 beads (Invitrogen) for 15 min, and then 0.6 μg purified PCNA-H6 was included in the mixture for another 40-min incubation. After thorough washing, the amounts of PCNA-H6 bound to the beads were analyzed by SDS-PAGE. (B) Three micrograms of BaMV gRNA was mixed with 200 μl oligo(dT)15 beads for 15 min, and then 3 μg PCNA-H6 variants (wild type or D41A or F114A mutant) was included in the incubation mixture as described above. PCNA-H6 bound to the beads after extensive washing was analyzed by SDS-PAGE. Arrows indicate the position of PCNA-H6 on gels.
DISCUSSION
PCNA, an evolutionarily conserved protein present in eukaryotic cells and archaea, plays an essential role in many biological processes such as DNA replication and repair and cell cycle control (22). Actually, PCNA molecules from different organisms are functionally interchangeable in the aspect of interacting with DNA polymerase δ (23, 24). PCNA acts as the sliding clamp, a trimeric structure with a pseudo-6-fold symmetry ring around DNA. During DNA synthesis, this trimeric structure serves to enhance the processivity of DNA polymerase δ/ε and serves as a platform for recruitment and dynamic exchange of other replication proteins (21). Furthermore, PCNA with monoubiquitin on the K164 residue recruits polymerases η, ι, and κ to the replication fork and activates translesion synthesis, a DNA damage tolerance mechanism allowing continued DNA synthesis in the presence of damaged DNA templates (29). Therefore, the expression level of PCNA correlates positively with the cell reproduction potential in eukaryotic cells. PCNA also functions in DNA repair through interactions with DNA polymerase δ/ε, RFC, DNA lig1, and Fen1 to promote DNA fragment resynthesis (20). In addition, PCNA interacts with MSH2/MSH6 heterodimer and stimulates the preferential binding of MSH2/MSH6 to the incorrectly paired DNA bases during the process of mismatch repair (30). The role of PCNA in cell cycle control involves p21, an inhibitory protein to the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes in DNA-damaged and aging cells. p21 strongly binds PCNA by means of its PCNA-interacting protein box, and this binding presumably blocks PCNA from interacting with RFC and DNA polymerase δ, thus stopping DNA synthesis (31). Arabidopsis PCNA was found to interact with human p21 (32); consequently, it has been suggested that PCNA is also involved in cell cycle control in plants. Besides these core functions, PCNA is thought to participate in many other physiological events, such as chromatin remodeling and epigenetics, as more and more PCNA-interacting proteins are discovered (21).
In this study, downregulation of PCNA in N. benthamiana via VIGS showed a negative effect on BaMV CP accumulation; nonetheless, additional expression of PCNA in protoplasts prepared from healthy leaves also suppressed the replication of BaMV. These apparently contradictory results can be attributed to the drastic effect of PCNA silencing on the development of newly emerged leaves, evidenced by the abnormal appearance of chloroplasts. Considering that BaMV viral RNA may target to chloroplasts for replication (33), changes in chloroplasts surely would affect BaMV replication. Accordingly, this adverse effect of PCNA silencing on BaMV replication was rather indirect, resulting from globally cellular changes. In comparison with the long-period protocol for VIGS, overexpression of PCNA in protoplasts was in a transient course, a condition more suitable for examining the direct effect of PCNA on BaMV replication.
Although PCNA is generally considered to be a nuclear protein, the coimmunoprecipitation assay in this study suggests that PCNA might associate with the BaMV replication complex solubilized from P30. Furthermore, the in vitro pulldown assay demonstrates a binding ability of PCNA to BaMV gRNA. Therefore, some PCNA molecules may remain in or relocalize into the cytoplasm to participate in the assembly of the viral replication complex. The in vitro RdRp activity assay confirmed that the addition of E. coli-expressed PCNA in P30 suspension suppressed the SF4-dependent RNA polymerization activity of REPBaMV. In summary, the data collected in this study strongly suggest a novel function of plant PCNA in preventing the replication of BaMV and some other potexviruses.
Mammalian genomes contain one copy of the PCNA gene despite the presence of PCNA pseudogenes in some species (34, 35). Crops, including but not limited to Oryza sativa, Glycine max, Brassica napus, and Pisum sativum also contain one PCNA copy in their genomes (22). Nonetheless, some plants such as A. thaliana and Zea mays have two PCNA isoforms, with 96.6 and 98.5% identities in amino acid sequence within each isoform pair, respectively. Two PCNA transcripts are found in N. benthamiana genomic and transcriptomic databases (http://benthgenome.qut.edu.au/) with slight nucleotide variations located in both regions of the 5′-untranslated region (UTR) and ORF. Variations within the open reading frames cause only two amino acid substitutions among 264 total amino acids, and both changes involve valine and isoleucine, suggesting that the two PCNA isoforms of N. benthamiana have the same biochemical activities. Although the differentially spatiotemporal expression of these two PCNA molecules is still unclear, the VIGS method used in this study practically could knock down both of them. The great amount of starch accumulated within chloroplast in the PCNA-silenced plants (Fig. 4) probably reflects that sucrose synthesized in the chloroplast could not be transported into the sink, where cell division was slowed down due to the downregulation of PCNA. Nonetheless, the mechanism underlying the abnormality of the chloroplast in response to PCNA silencing may be more complicated, hence deserving further study.
PCNA was originally identified as an antigen that is expressed in the nuclei of cells during the S phase of the cell cycle. However, recent data indicate that PCNA may perform quite different roles outside the nucleus. A high level of PCNA accumulates exclusively in the cytosol of mature neutrophils, and these PCNA molecules constitutively associate with procaspases, thus preventing neutrophils from apoptosis (36). A chromosome region maintenance 1 (CRM1)-dependent nuclear-to-cytoplasmic relocalization during granulocytic differentiation is responsible for the accumulation of PCNA in cytosol of mature neutrophils (37). Furthermore, PCNA was also found in association with caspase-9 in the cytosol of the SH-SY5Y neuroblastoma cell line; moreover, S-nitrosylation of PCNA at the residues C81 and C162 decreases this interaction, leading to caspase-9 activation (38). PCNA is overexpressed in cancer cells. The interaction of cell-surface-presenting PCNA with the cytotoxicity receptor NKp44 of natural killer (NK) cells inhibits NK-mediated cell lysis and IFN-γ secretion (39). Therefore, it has been thought that cancer cells exploit PCNA to evade the attack of NK cells. Compared to increasing evidence showing the functional versatility of mammalian PCNA, whether plant PCNA moonlights outside the nucleus is still uncertain. Recently, a biomolecular fluorescence complementation assay indicated that PCNA1 and PCNA2 of A. thaliana probably form homo- and heterotrimeric complexes in the nucleus as well as in the cytoplasm (26). Therefore, it is highly possible that some PCNA molecules are present in the cytoplasm of N. benthamiana cells, where they have a chance to interact with the BaMV replication complex, consequently downregulating the replication efficiency of the virus. If this is the case, the mechanism by which PCNA stays in the cytoplasm or relocalizes from the nucleus to the cytoplasm in plant cells represents an interesting future inquiry.
The involvement of PCNA in virus replication has been reported in a couple of DNA viruses. Geminiviruses replicate in the nuclei of their plant hosts by recruiting host replication machinery because they lack the DNA polymerase-encoding gene in their own single-stranded DNA genomes. Rep protein of Indian mung bean yellow mosaic virus (IMYMV), a bipartite begomovirus from the family Geminiviridae, has a binding affinity to pea PCNA. The Rep-PCNA interaction impairs the site-specific nicking-closing activity and the ATPase function of IMYMV Rep that are required for viral replication (40). Sulfolobus islandicus rod-shaped virus 2 (SIRV2), which infects hyperthermophilic archaea, does not encode its own DNA polymerases. Five SIRV2 proteins were found to interact with the heterotrimeric Sulfolobus solfataricus sliding clamp (SsoPCNA-1 to -3), through which the host replication machinery may be recruited for the virus DNA replication (41). Human PCNA is loaded onto the newly synthesized DNA of Epstein-Barr virus during lytic replication, and the loading might trigger transfer of a series of host mismatch repair proteins, such as MSH2 and MSH6, to the sites of viral DNA synthesis (42). This recruitment is important for the repair of mismatches arising during viral replication. As for plant RNA viruses, PCNA was found to be one of the yeast proteins that interacted with the p33 replication protein of TBSV in a yeast proteome microarray assay (43). Moreover, overexpression of PCNA increased the accumulation of a TBSV replicon RNA in Saccharomyces cerevisiae (44). In this study, we found that PCNA of N. benthamiana has a binding affinity to the genomic RNA of BaMV. This PCNA-RNA interaction might impose an inhibition on the replication of BaMV. To our knowledge, this is the first report of PCNA being able to suppress the replication of a plant RNA virus. The crucial molecular features or sequence motifs on BaMV gRNA and satBaMV SF4 that support recognition by plant PCNA will be investigated in the future. Furthermore, whether BaMV-encoded proteins, including REPBaMV itself, have binding affinities directly to PCNA will be an interesting query in the future.
It has been extensively observed that BaMV infection hardly occurs in the young leaves of N. benthamiana. It seems that the systemic spreading of BaMV from infected tissues into newly emerged leaves is restricted. A comparable phenomenon has been described in plants infected by other plant viruses. For example, Potato virus X (PVX) cannot invade the meristem of its hosts. The endogenous RNA-dependent RNA polymerase RDR6 was ascribed as a factor for this meristem exclusion in N. benthamiana (45). RDR6 was proposed to recruit a PVX-derived silencing signal to trigger an immediate silencing response against the virus as it enters the meristem. This study suggests that PCNA may provide an additional control to suppress replication of PVX in the rapidly growing tissue. Presumably, RDR6 also plays a role in preventing the newly emerged leaves of N. benthamiana from being invaded by BaMV. Besides, other cellular factors may take part in this restriction. This study used a transgenic N. benthamiana line to examine the expression profiles of PCNA and the recombinant BaMV-encoded proteins, including GFP and CP. The profiles of PCNA and the viral proteins were explicitly opposite (Fig. 8). The viral proteins were found mostly in relatively older leaves where PCNA was scarce; in contrast, the viral proteins were almost invisible in newly emerged leaves where PCNA was abundant. Since all cells of the entire transgenic plant contained the cDNA copy of BaMV-GFP, any mechanism that imposed a restriction on BaMV spreading was not sufficiently accountable for the absence of the virus in the newly emerged leaves. Mechanisms working at the single-cell level must have a role in this regard. That the enhanced PCNA level in the very young leaves suppressed the transcription from the 35S promoter in BaMV-GFP transgenic plants, thus reducing the GFP signal below detection level, could be a possible explanation. Nonetheless, overexpression of PCNA in protoplasts inhibited the accumulation of BaMV CP even when BaMV gRNA was used in transfection (Fig. 5C), suggesting that the inhibition can be devoid of the 35S promoter. Moreover, addition of PCNA to the in vitro BaMV RdRp reaction mixture decreased the production of RNA from SF4 template (Fig. 9). Collectively, it is very likely that PCNA plays a direct inhibitory role in newly emerged leaves by creating a sterile area where BaMV hardly replicates. PCNA also suppresses replication of FoMV and PVX according to the protoplast experiments (Fig. 6). Thus, it will be an interesting query to survey the effects of PCNA on the replication of diverse plant RNA viruses, given the findings that PCNA was an inhibitory factor against potexviruses but an upregulator for a TBSV replicon RNA (44).
MATERIALS AND METHODS
Plasmids.Binary plasmids pERep-HA and pKSF4 were used to produce the hemagglutinin tag-fused REPBaMV (REPBaMV-HA) and the satBaMV SF4 variant, respectively, in N. benthamiana as in previous reports (9, 10). The cDNA encoding N. benthamiana PCNA was cloned by PCR from a leaf cDNA preparation according to the transcriptomic information available in the N. benthamiana Genome and Transcriptome Sequencing Consortium website (http://benthgenome.qut.edu.au/). Plasmid pBI-PCNA for the production of extra PCNA in protoplasts was constructed by positioning the PCNA cDNA downstream of the Cauliflower mosaic virus (CaMV) 35S promoter in pBI221 using the restriction sites SmaI and SacI. A 6×His tag-coding sequence was fused in frame to the 3′ end of the PCNA cDNA in pBI221-PCNA according to the QuikChange site-directed mutagenesis protocol. The resulting plasmid, pBI-PCNA-H6, was used to confirm the production of PCNA in protoplasts attributed to the transfected plasmids. Mutant pBI-PCNA, which produced PCNA(D41A) or PCNA(F114A) in protoplasts, was created also by the QuikChange site-directed mutagenesis protocol. PCNA cDNA was inserted into pEpyon plasmid to become pE-PCNA so that PCNA could be expressed transiently in plants through agroinfiltration. To produce PCNA in Escherichia coli, the PCNA cDNA was inserted into the pET29 plasmid via restriction sites NdeI and XhoI. The resulting plasmid, pET29-PCNA, was used to produce a C-terminally His6-tagged PCNA (PCNA-H6) in E. coli BL21(DE3) cells. The mutant PCNA cDNA carrying the desired mutation for D41A or F114A substitution was grafted from the pBI221-based plasmids to pET29 so that the mutant PCNA could be produced also in E. coli cells. A fragment of the PCNA cDNA, ranging from nucleotides 236 to 477, was amplified by PCR and inserted into pTRV2 plasmid via restriction sites EcoRI and XhoI. The resulting plasmid, pTRV2-PCNA, was used together with pTRV1 via agroinfiltration to silence the expression of PCNA in N. benthamiana. pTRV2-Luc and pTRV2-PDS, which carry partial nucleotide sequences of luciferase and phytoene desaturase genes, respectively, served as the controls in the Tobacco rattle virus-induced gene silencing experiments. An infectious clone pCBG, containing a duplicated CP promoter-driven GFP gene, enabled us to monitor the replication and systemic movement of BaMV in host plants based on the expression of GFP (3). In this study, pCBG and pCF were used to establish the infection of BaMV and Foxtail mosaic virus (FoMV) in protoplasts, respectively (4, 10). pXTAL, an infectious clone of Potato virus X (PVX), was constructed by substituting the BaMV cDNA in BaMV infectious clone with the cDNA prepared from a local PVX strain (GenBank accession no. AF272736).
Agrobacterium infiltration.The recombinant Agrobacterium tumefaciens C58C1 strain harboring pERep-HA or pKSF4 was harvested from a 2-day-old LB culture and suspended in buffer of 10 mM MES (morpholineethanesulfonic acid [pH 5.5]) and 10 mM MgCl2 to an optical density at 600 nm (OD600) of 0.5. The two strains were equally mixed and infiltrated into the undersides of leaves of 4-week-old N. benthamiana. The leaves harvested 2 days after agroinfiltration were used to prepare the REPBaMV-containing membrane fraction. In virus-induced gene silencing (VIGS) experiments, the mixed suspension of Agrobacterium strains harboring pTRV1 and the specified pTRV2 derivative was infiltrated into N. benthamiana leaves per the protocol described above. The appearance of white spots on the leaves of plants that received pTRV1 and pTRV2-PDS at about 4 to 5 weeks after agroinfiltration indicated success of silencing treatments.
Identification of potential host factors.N. benthamiana leaves agroinfiltrated with pKSF4 and pERep-HA, as described above, were homogenized in buffer containing 50 mM Tris (pH 8.0), 10 mM KCl, 15 mM MgCl2, 0.1% (vol/vol) β-mercaptoethanol, 20% (vol/vol) glycerol, and 1× complete EDTA-free protease inhibitor cocktail (Roche). After centrifuging at 800 × g for 10 min, the supernatant was centrifuged again at 30,000 × g for 35 min. The pellet (P30 [0.1 g]) was washed twice with the same homogenization buffer plus 1% Triton X-100 and subsequently suspended in 200 μl 75 mM NaCl plus 7.5 mM sodium citrate solution. An aliquot (180 μl) of the P30 suspension was mixed with 60 μl 4× sample buffer (160 mM Tris [pH 8.0], 16% glycerol, 20% β-mercaptoethanol, 0.04% bromophenol blue, and 4 M urea) plus 5 μl protease inhibitor cocktail for 1 h. The mixture was centrifuged again at 30,000 × g for 35 min, and the supernatant was subjected to SDS-PAGE (4 to 13% polyacrylamide) following incubation at 60°C for 30 min. The P30 sample prepared from leaves agroinfiltrated with only pKSF4 was analyzed in parallel as the control. The gel slice representing the REPBaMV-associated protein complex, according to the image of the Western blot and the relative migration compared to protein standards, was cut from the polyacrylamide gel, and the proteins within were identified by tandem mass spectrometry using an Applied Biosystems QStar liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrometer (Life Technologies Corp., Carlsbad, CA, USA) with Mascot software using the NCBI nonredundant database. The important parameter settings for Mascot analysis were as follows: mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance, ±0.5 Da; and maximal missed cleavages, 2.
Leaf inoculation.Proliferation of BaMV in N. benthamiana plantlets was estimated by evenly smearing a suspension of 0.5 μg virions in 10 μl water onto the surface of a leaf with the assistance of carborundum dust. Two leaves were inoculated per plantlet. The dust on the leaves was washed off 1 day after inoculation, and the leaves were collected 4 days after inoculation for further analysis.
Protoplast transfection.Proliferation of BaMV in N. benthamiana protoplasts was estimated by gently mixing 1 × 105 protoplasts, prepared from 5-week-old N. benthamiana leaves, with pCBG and the indicated plasmids in the presence of 20% polyethylene glycol 4000 (PEG 4000) in a 2-ml round-bottom vial for 30 min as described previously (18). The transfected protoplasts were then cultivated at room temperature, under a constant light, in growth buffer (0.55 M mannitol-MES [pH 5.7], 1 μM CuSO4, 1 μM KI, 1 mM MgSO4, 0.2 mM K2HPO4, 1 mM KNO3, 10 mM CaCl2, and 30 μg/ml cefotaxime) for 20 h. Viral RNAs and virus-encoded proteins accumulated in protoplasts were analyzed by Northern and Western blotting, respectively. Infection of FoMV and PVX in protoplasts for the transfection step was done by using pCF and pXTAL, respectively. The rest of the procedures were the same as described above.
In vitro RdRp activity assay.The assay for BaMV genome synthesis in vitro was performed using the P30 membrane fraction extracted from BaMV-infected leaves 5 days postinfection. The P30 isolated from 5 g leaves was thoroughly suspended in 1.5 ml TG buffer (50 mM Tris [pH 8.8], 2.5% (vol/vol) glycerol, 10 mM NaCl, and 1× complete EDTA-free protease), supplemented with 1.5% NP-40, 0.3% Sarkosyl, and 0.05% or 0.1% SDS as indicated. After centrifugation for 30 min at 30,000 × g, 25 μl of the supernatant was mixed with 7 μl RdRp reaction buffer (150 mM Tris-HCl [pH 8.8], 50 mM MgCl2, 250 mM NaCl, 100 mM dithiothreitol [DTT], 10 mM ATP, 10 mM CTP, 10 mM GTP, 10 mM UTP), 1.5 μl [α-32P]UTP (6,000 Ci/mmol) (Perkin Elmer), and 1.5 μl RNase inhibitor (Roche). The reaction mixture was kept at 26°C for 3 h, and the reaction mixture was subsequently electrophoresed on a 1% agarose gel. The radiolabeled RNA products were visualized with a phosphorimager (Fujifilm BAS-2500).
To examine the effect of E. coli-expressed PCNA on SF4 RNA-dependent polymerase activity, the P30 fraction prepared from leaves that had been infiltrated with A. tumefaciens strains carrying pERep-HA and pKSF4 plasmids for 2 days was utilized. An aliquot of the P30 suspension in TG buffer supplemented with 0.3% Sarkosyl was mixed with the following: RdRp reaction buffer, [α-32P]UTP, RNase inhibitor, along with specified amounts of purified PCNA variants (wild type [WT], D41A, or F114A) or bovine serum albumin (BSA) to create a final 35-μl volume solution. The reaction conditions and visualization of the radiolabeled products were the same as described above.
Coimmunoprecipitation assay.N. benthamiana leaves were agroinfiltrated with pERep-HA for the transient expression of REPBaMV-HA and pKSF4 for the transient expression of satBaMV SF4; both utilized the same method described above. PCNA or OFP was coexpressed in leaves by agroinfiltration with pE-PCNA or pE-OFP, respectively. The P30 fraction extracted from the leaves 2 days post-agroinfiltration was incubated overnight in TG buffer supplemented with 0.3% Sarkosyl. After centrifugation for 10 min at 30,000 × g, a 50-μl aliquot of the supernatant was mixed with a 3-μl antibody that specifically recognizes HA tag, PCNA, or His tag. The total aliquot and antibody solution was combined with 5 μl protein G Mag Sepharose Xtra beads (GE Healthcare). The bulk solution was removed by a magnet attracting the beads. Afterwards, the beads were washed three times with 500 μl TG buffer supplemented with 0.3% Sarkosyl. The proteins still bound to the beads were then analyzed by Western blotting using the specified antibody.
Preparation of recombinant PCNA.E. coli BL21(DE3) cells harboring pET29-PCNA or the indicated mutant plasmids were aerobically cultivated at 37°C in LB. IPTG (isopropyl-β-d-thiogalactopyranoside [1 mM]) was added into the broth when the OD600 reached 0.8, and the cells were further cultivated at 28°C for 5 h. The pelleted cells were suspended in extraction buffer (250 mM Tri-HCl [pH 8.0], 200 mM NaCl, and 10% glycerol) and disrupted by sonication. The clarified supernatant was loaded onto a 5-ml Ni2+-nitrilotriacetic acid (NTA) column, and the C-terminally His6-fused PCNA was finally eluted with the extraction buffer supplemented with 500 mM imidazole. The recombinant PCNA was further purified with a Sephacryl S300HR 10/60 column.
Preparation of BaMV gRNA.BaMV virions were isolated from the virus-infected leaves of N. benthamiana through steps of PEG 6000 precipitation and sucrose centrifugation as described in the previous report (46). The purified virions were heated at 60°C in buffer that contained 6 mM phosphate (pH 7.2), 0.2 mM EDTA, 1% β-mercaptoethanol, 1% bentonite, and 1% SDS for 5 min. The viral gRNA was then purified via phenol extraction and ethanol precipitation.
Pulldown assay.An indicated amount of BaMV gRNA was mixed with 200 μl Dynabeads oligo(dT)15 suspension (Invitrogen) and incubated at room temperature for 15 min. The purified PCNA variants (each 0.6 μg) were then included, and the mixture was incubated for another 40 min. After extensive washing with buffer (25 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5% glycerol), the beads were suspended in an aliquot of 1× protein sample buffer and heated at 95˚C for 5 min. The amount of PCNA that associated with the beads was then analyzed by SDS-PAGE.
Transmission electron microscopy.Leaves from the targeted gene-silenced N. benthamiana were collected and cut into pieces of approximately 1 mm2. The sample was treated with 2.5% glutaraldehyde, followed by dehydration. It was then embedded into LR white resin (London Resin Company), and the resin was sliced into 60-nm-thick sections. The sections were then treated consecutively with 2% uranyl acetate for 20 min and 0.5% lead citrate for 10 min and observed using a JEOL JEM-1400 transmission electron microscope.
RNA analysis.To prepare real-time qPCR analysis, the leaf RNA was isolated using TriSolution Plus reagent (GeneMark, Taiwan) and primed to synthesize cDNA using an oligo(dT)15 primer and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Fitchburg, WI). Each 15-μl qPCR mixture contained 4 μl cDNA, 7.5 μl KAPA Sybr Fast qPCR master mix (Kapa Biosystems, Wilmington, MA), and the paired primers (each 0.13 μM) specific for detecting the transcripts of PCNA (5′-TGGAATTACGGCTTGTTCAG-3′ and 5′-TTGAGCATTTTAGCCATGTTA-3′) and the internal control β-actin (5′-GATGAAGATACTCACAGAAAGA-3′ and 5′-GTGGTTTCATGAATGCCAGCA-3′). The cycling condition began with an initial hold at 95°C for 3 min, followed by 30 cycles of 3 s at 95°C, 20 s at 58°C, and 20 s at 72°C. Reactions, including data collection, were carried out with the TOptical Gradient 96 real-time thermocycler (Biometra GmbH, Germany). For Northern blotting analysis, each RNA sample was treated with 1.2 M glyoxal-containing 10 mM phosphate buffer [pH 6.3] and separated on a 0.8% agarose gel. After transfer and fixation onto a nylon membrane, the samples were hybridized with a 32P-labeled probe complementary to the 3′-UTR of BaMV as previously described (10).
Protein analysis.Proteins extracted from plants or protoplasts were separated by standard 12% SDS-PAGE, followed by Coomassie blue staining, silver staining, or Western blotting assays using indicated rabbit antibody for specific protein recognition. Goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody was used as the secondary antibody, accompanied with Immobilon Western chemiluminescent HRP substrate (Millipore) in the Western blot. The large subunit of RuBisCO (RbcL) shown in the staining gels was used as an internal control for normalization whenever the expression of targeted protein needed to be relatively compared.
ACKNOWLEDGMENTS
This work was supported by grants from MOST 106-2313-B-005-021-MY3, Ministry of Science and Technology, Taiwan, ROC.
There is no conflict of interest regarding this study.
FOOTNOTES
- Received 11 June 2019.
- Accepted 30 August 2019.
- Accepted manuscript posted online 11 September 2019.
- Copyright © 2019 American Society for Microbiology.
