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Chinese Science Bulletin 2005 Vol. 50 No. 4 305—310

Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus ZHANG Lingdi, WANG Zhaohui, WANG Xianbing, LI Dawei, HAN Chenggui, ZHAI Yafeng & YU Jialin State Key Laboratory for Agrobiotechnology, China Agricultural Univer-sity, Beijing 100094, China Correspondence should be addressed to Yu Jialin (e-mail: bnyvv@public. bta.net.cn)

Abstract Functional analysis for gene silencing suppres-sor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- in-filtration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by ho-mologous sequence was strongly suppressed by the immix-ture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results sug-gested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors. Keywords: Beet necrotic yellow vein virus P14, post-transcriptional gene silencing, Rice black streak dwarf virus S6, suppressor.

DOI: 10.1360/982004-731

RNA silencing, also named post-transcriptional gene silencing (PTGS), is a process of RNA degradation widely occurring in living organisms. This mechanism was in-volved in the recognition of invaded foreign DNA, stable gene expression and virus defense. Nevertheless, it was revealed that specific proteins were encoded by some plant viruses for suppression of gene silencing induced by the virus infection, representing a conflict between the plant defense system and the viruses directed PTGS sup-pression. Since the first silencing suppressor of HC-Pro gene in Potato virus Y (PVY) was reported[1], many dif-ferent suppressor proteins were identified in plant vi-ruses[2—6]. Due to the different functions and structural characters of the suppressors, the virus infections were varied from the symptoms to the movement in host plant and replications. The cystine-rich motifs were found in many virus-encoded suppressors reported previously[7], in which a zinc-finger structure was related with the nucleo-tide binding activity as found in P15 protein of Peanut

clump virus (PCV)[8] and γb protein of Barley stripe mo-saic virus (BSMV)[9]. Analysis of virus-encoded suppres-sors would provide the evidence for identification of the cell proteins related with PTGS and understanding of the mechanism of gene silencing.

The 14 kD protein (P14) encoded by Beet necrotic yellow vein virus (BNYVV) RNA2 is a cystine-rich pro-tein that could bind with zinc in vitro[10]. The accumula-tion level of BNYVV RNA and coat protein would be reduced by mutation of P14[11]. Since BNYVV could move from cell-to-cell in the host plant when its coat pro-tein was mutated[12], it was suggested that the P14 possibly bond with the viral RNA in vivo as a molecule-mate to protect virus and enhance its movement through the host cells. On the other hand, although function of the S6 pro-tein encoded by Rice black-streaked stunt virus (RBSDV)

RNA segment 6[13] is unknown, the prediction by com-puter analysis and comparison with the databases revealed that the S6 protein had a highly conserved hydrophilic structure and contained many cross-membrane motifs that were frequently involved with the activities of RNA or ATPase bindings. These results from previous reports perhaps give us a hint that the P14 gene of BNYVV RNA2 and the S6 gene of RBSDV may code for sup-pressing proteins against plant gene silencing.

In this study, a Potato virus X (PVX) vector and the agro-infiltration method [14] were employed for infection of N. benthamiana containing a GFP transgene. By obser-vation of the phenotypes and molecular detection of the inoculated plants, the P14 gene of BNYVV RNA2 and the S6 gene of RBSDV were analyzed for their functions as silencing suppressors.

1 Materials and methods

(ⅰ) Bacteria, plants, viruses and cDNA clones used in the research. Escherichia coli strain DH10B, Agro-bacterium tumefaciens strain GV3101 and the full-length cDNA clone pFny209 of Cucumber mosaic virus (CMV) RNA2 were the stocks of our group. The binary vectors pBINmGFP5 and pGreen208 for expression of GFP or full-length PVX RNA in plants respectively (Fig. 1), and the pure line 16C of N. benthamiana containing a gfp transgene were kindly provided by Dr. Baulcombe D. C. at Sainsbury laboratory, John Innes Center, UK. BNYVV was isolated from Huhhot of Inner Mongolia and propa-gated on Tetragonia expensa for virus purification, while RBSDV was extracted directly from the diseased maize leaves collected in the fields of Tianmen, Hubei Province.

(ⅱ) Construction of binary vectors for plant infiltra-tion. On the template of pGreen208, a primer pair of ND108-F corresponding to 5611—5625 nt of the PVX sequence [15] fused with a multiple-cloning site of MluⅠ- ClaⅠ-SmaⅠ-NotⅠ-SalⅠ (5′-TTTTTTACGCGTATC- GATCCCGGGCGGCCGCGTCGACCGCCGATGAAC- GGTT-3′), and ND108-R complementary to 5645—5660

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nt of the PVX sequence [15] fused with restriction digestion sites of MluⅠ and NheⅠ (5′-TTTTTTACGCGTGCT- AGCTGGTGCTGACCTCTTT-3′) were used for a reverse PCR amplification. Having been treated with DpnⅠ to remove the template DNA, the PCR product was digested with MluⅠ for re-ligation to form the circler plasmid pND108. By nucleotide sequencing from both directions, it was confirmed that, in pND108, the gfp fragment was deleted and the multiple-cloning site of MluⅠ-ClaⅠ- SmaⅠ-NotⅠ-SalⅠ was inserted correctly.

(ⅲ ) Agro-infiltration of plants [14]. The bacteria transformed by the recombinant plasmids were cultured in LB medium (containing 100 mg/L Kan, 2 mg/L Tet, 10 mmol/L MES and 20 µmol/L Acetosyringone) by shaking at 28 overnight. After centrifugation at 6000 r℃ /mim for 5 min, the pellet was re-suspended in 10 mmol/L MgCl2, 10 mmol/L MES and 100 µmol/L Acetosyringone to a final density of A600 = 0.7 and incubated at room tempera-ture for more than 3 h. Six to eight plants of N. bentha-miana were infiltrated on their leaves by syringe injection for each treatment. The plants inoculated with different vectors were assayed for the variations of their phenotypes and gfp gene expression. Plant green fluorescence was observed under a long-wave UV light (B-100AP/R, UVP) and recorded by a digital camera (CoolPix 4500, Nikon) with an orange filter (Gelatin filter No. 15, Kodak).

RNA template was extracted from the leaves of T. expensa infected by BNYVV and used for RT-PCR with a primer pair of Bny14-1 corresponding to 4033—4056 nt of BNYVV RNA2 sequence [16] containing a MluⅠ site (5′-AAGGCACGCGTAGAATGAGTATGGGGATGGTA-GAT-3′), and Bny14-2 complementary to 4403—4425 nt of BNYVV RNA2 sequence [16] containing a SmaⅠ site (5′-CCCGCCCCGGGTTACACCTCAGGATCGACAAT-

(ⅳ) Nucleic acids hybridization. Total RNA was extracted from N. benthamiana by SDS method[19] and measured by means of spectral analysis. After separation on 1.2% agarose gel denatured with 0.1% formaldehyde, 10 μg RNA was transferred onto nylon membrane (Hy-bond N+, Amersham) with 20×SSC buffer. The cDNA probes of GFP or PVX coat protein were labeled by ran-dom primers used for Northern blot and the results were recorded by PhosphorImager (Storm 820, Molecular Dy-namic). For Southern blot, total DNA was extracted from N. benthamiana by the CTAB method and measured by means of spectral analysis. Twenty micrograms of DNA were digested by HaeⅢ and transferred onto nylon membrane after separation by electrophoresis. Probe preparation and hybridization were carried out same as described above.

AA-3′). Through the intermediate procedures, the ampli-fied product was introduced into pND108 to create the plant expression vector pND14K. Similarly, viral RNA from maize leaves infected by RBSDV was used for RT-PCR amplification with primers of PS6-1 correspond-ing to 1—21 nt of RBSDV S6 sequence (5′-AAGTTTT- TTGAGTCTGAGATAC-3′) [13] and PS6-2 complemen-tary to 2626—2645 nt of RBSDV sequence (5′-GACAT- CAGCTGATTTGAGTC-3′)[13]. By ligation with pND108, the vector pNDS6 was created for plant expression. In parallel, the CMV 2b gene was amplified by PCR on the cDNA template of pFny209 and a pair of primers Fny2B-1 corresponding to 2419—2438 nt of CMV RNA2 (5′- ATGGAATTGAACGTAGGTGC-3′) [17,18], and Fny2B- 2 complementary to 2732—2751 nt of the CMV RNA[17,18] (5′-TCAGAAAGCACCTTCCGCCC-3′). The amplified product was ligated into plasmid pND108 and selected for the recombinant vector of pND2B. All plasmids made above were transformed into A. tumefaciens strain GV3101 to be used for plant infiltration.

2 Results (ⅰ) Construction of binary vectors for plant expres-

sion. The vectors provided by Dr. Baulcombe D. C. or the derivates from pGreen208 are shown in Fig. 1, which will be used for transient expression in plant cells medi-ated by agro-infiltration.

Fig. 1. Scheme of the expression vector constructions. X represents the genes of BSDV S6, BNYVV P14 or CMV 2b, respectively.

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(ⅱ) The phenotypes of N. benthamiana-16C infil-trated by the binary vectors containing P14 or S6 gene. To induce gene silencing in transgenic N. benthamiana- 16C, the binary vectors containing gfp gene (pBINmGFP5 or pGreen208) were used for plant infiltrations mediated by Agrobaterium. The agrobacteria carrying PVX vectors of pND14K or pNDS6 were mixed with plasmid pBINmGFP5 in the same density (A600) for plant inocula-tion. From 5—19 days post inoculation (dpi), the pheno-types of gene silencing on the infiltrated plants were ob-served under UV light in each 2 d. Compared with the plants that were inoculated with pBINmGFP5 alone or pBINmGFP5 plus pND108 (Fig. 2(b) and (c)), the red colors on young leaves of the plants that were inoculated simultaneously with pND14K or pNDS6 appeared 2—3 d later than the controls. By the treatment, the red colors were limited locally on the first to second leaf up to the inoculated site (Fig. 2(d) and (e)) while other parts of the plants were kept in green as the negative control (Fig. 2(a)), similar to that by pND2B infection (CMV 2b gene)(Fig. 2(f)). Among these, the plants infected by pBINmGFP5 plus pND14K showed red along the veins on the top leaves (Fig. 2(e)), while those injected with pBINmGFP5 plus pNDS6 had smaller red mottles (Fig. 2(d)).

By Northern blot, results showed that the accumula-tion levels of gfp mRNA in the host plants were coincident with their phenotypes by fluorescence assay (Fig. 3). In the plants inoculated with pBINmGFP5 plus pNDS6 or pND14K (Lanes 3 and 5), the accumulation of gfp mRNA

was slightly lower than that injected with pNDS6 or pND14K alone (Lanes 2 and 4), but still kept a relative high level as that infected with pND2B (Lanes 6 and 7). However, in the control plants infiltrated with vectors of pBINmGFP5, pBINmGFP5 plus pND108 or pGreen208 containing the gfp gene only, no gfp mRNA was detected (Lanes 1, 8 and 9). These results revealed that, during the process of gfp mRNA degradation induced by the ho-mologous sequence, expression of BNYVV P14 gene or RBSDV S6 gene had interfered with the mechanism of gene silencing in the transgenic N. benthamiana-16C, suggesting that the function of suppressing to gene silenc-ing is similar to that of CMV 2b [18].

(ⅲ) DNA methylation detection of N. benthamiana- 16C infiltrated by the binary vectors containing P14 or S6 gene. In order to assay genomic DNA methylation, total DNA was extracted from the plants at 60 dpi. After re-striction digestion with HaeⅢ and separation on 1.8% agarose gel, the DNA samples were transferred to a nylon membrane and hybridized with a cDNA probe of full- length gfp gene (Fig. 4). In the repeated experiments with different individual plants, only small digested DNA fragments were probed in the plants that were inoculated with pBINmGFP5 plus pNDS6 (Lane 2). In contrast, in the plants infiltrated synchronously by pBINmGFP5 plus pND2B (Lane 3) or pBINmGFP5 plus pND14K (Lane 4), the gfp DNA was partially degraded into small fragments, indicating a lower level of gfp DNA methylation than that in plants infected by pGreen208 alone (Lane 5). Coinci-dently with the gfp expression of N. benthamiana-16C in

Fig. 2. The phenotypes of N. benthamiana-16C inoculated by binary vectors. The plants were inoculated with A. tumefaciens strain GV3101 alone (a), pBINmGFP5 (b), pBINmGFP5 plus pND108 (c), pBINmGFP5 plus pNDS6 (d), pBINmGFP5 plus pND14K (e) and pBINmGFP5 plus pND2B (f). Result was recoded at 19 dpi.

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Fig. 3. Northern blot of gfp mRNA in N. benthamiana-16C inoculated by binary vectors. The plants were inoculated with pBINmGFP5 plus pND108 (Lane 1), pNDS6 (Lane 2), pBINmGFP5 plus pNDS6 (Lane 3), pND14K (Lane 4), pBINmGFP5 plus pND14K (Lane 5), pND2B (Lane 6), pBINmGFP5 plus pND2B (Lane 7), pBINmGFP5 (Lane 8) and pGreen208 (Lane 9). Samples were collected at 19 dpi.

Fig. 4. Detection of gfp DNA methylation in N. benthamiana-16C in-oculated by binary vectors. The plants were inoculated with Agrobacte-rium tumefaciens strain GV3101 alone (Lane 1), pBINmGFP5 plus pNDS6 (Lane 2), pBINmGFP5 plus pND2B (Lane 3), pBINmGFP5 plus pND14K (Lane 4) and pGreen208 (Lane 5). Samples were collected at 60 dpi.

section (ⅱ), this result provided further evidence to prove that BNYVV P14 and RBSDV S6 had functions of gene silencing suppressor as that of CMV 2b protein [17], espe-cially the RBSDV S6 expression strongly interfering with DNA methylation in host plants.

(ⅳ) Symptom variations of plants infected by PVX vector and the P14 or S6 gene. Because of infection by PVX vectors carrying foreign genes, typical viral symp-toms appeared on the host plants after the infiltrations. Nevertheless, when a virus encoded factor was carried by the PVX vector, interference with the defense mechanism was displayed significantly with the variable symptoms on the host plants. After infiltrations of wild type N. bentha-miana with the PVX vector (pGreen208 alone or pBINmGFP5 plus pND108), or simultaneously with the recombinant PVX vectors containing the genes of RBSDV S6 or BNYVV P14 (pNDX, Fig. 1), the symptom varia-tions were observed at 9 dpi and virus replications were detected by Northern blot. The results showed that the symptoms became more severe on the plants co-infiltrated with pNDS6 or pND14K than those with PVX vector alone (Fig. 5), although typical infection phenotypes ap-peared on all plants inoculated with any PVX vectors. With pND14K or pNDS6, the young leaves of infected N. benthamiana were curled and wilted to death (Fig. 5(c) and (d)), similar to those caused by inoculation with CMV 2b gene (pND2B) that was known as a silencing suppres-sor (Fig. 5(e)). In contrast, the plants infiltrated with PVX alone (pGreen208 or pBINmGFP5 plus pND108) showed mosaic and distorted symptoms, but the new leaves did

Fig. 5. The symptoms on infected N. benthamiana. The plants were inoculated with pBINmGFP5 (a), pGreen208 (or pBINmGFP5 plus pND108)(b), pBINmGFP5 plus pND14K (c), pBINmGFP5 plus pNDS6 (d) and pBINmGFP5 plus pND2B (e). Result was recoded at 9 dpi.

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not develop to death (Fig. 5(b)). With this result, although we cannot fully explain the functions of BNYVV P14 and RBSDV S6 proteins, it is suggested that these two vi-rus-encoded genes might be involved in the interference with the host defense system, mostly as the suppressors of gene silencing. With specific probe, Northern blot showed that the BNYVV P14 gene and RBSDV S6 gene inserted in the PVX vector were transcribed normally simultane-ously with expression of the PVX genome, which could move from cell to cell in host plants (data not shown). 3 Discussion

The mechanism of gene silencing in eukaryotes is involved in the defense system against virus invading and foreign nucleic acids, which has been established and de-veloped gradually during long term evolution as an im-mune system on the genomic level[20]. Facing the mecha-nism, the strategy of high frequency variation was adopted by virus to adapt or overcome the defense system, includ-ing the mutation of specific genes responsible for patho-genicity and genome replication. For example CMV 2b gene is an example of suppressors to host gene silenc-ing[21]. Due to the diversity of defense mechanisms, with which plants showed resistance to virus infection in dif-ferent levels, the strategies used by viruses in conflicting with gene silencing are various from the sequences and structures of suppressors, which function at the different points of PTGS regulation including silencing initiation, systemic signal transduction and silencing mainte-nance[22,23].

In this research, the binary vector pBINmGFP5 car-rying a gfp gene and the PVX expression vectors carrying a heterogenous viral gene (pNDS6 or pND14K) were used for simultaneous infiltration of gfp transgene N. bentha-miana-16C. The phenotypes showed that the systemic gfp gene silencing induced by pBINmGFP5 was not com-pletely suppressed by the participation of pNDS6 or pND14K, but by which the spread-off of gene silencing was significantly delayed. Since the silenced cells were blocked within a limited leaf area (Fig. 2(d) and (e)) and the gfp mRNA accumulation level were retained stably (Fig. 3), it was suggested that the BNYVV 14K and RBSDV S6 genes did not affect the production of gfp siRNA (date not shown), but the courses of gene silencing were restricted. As a result, these two plant virus genes did not function at the steps during gene silencing initiation, but they were related with silencing spread-off and main-tenance[18].

Since the target sequence in plant genomic DNAs would be methylated by introducing homogenous dsRNA[24], gene silencing could be induced in transcrip-tion stage (TGS) or post-transcription stage (PTGS) due to the functional characters of the silenced DNA sequences (promoter or coding region). Compared with the inherited characteristic of TGS, the red color phenotype of N. ben-

thamiana-16C infiltrated with pBINmGFP5 was not re-tained? in the progenies, indicating a PTGS reaction (data not shown). Fig. 4 shows that the methylation of the gfp DNA was completely suppressed by RBSDV S6 gene, but partially inhibited by BNYVV 14K gene at a similar level as that caused by CMV 2b gene, which interfered with methylation in nuclei[18]. Advanced research approaches showed that the chromosome methylation was closely related with an RNAi effector complex (RITS), a pro-tein-siRNAs complex induced by abnormal RNAs[25]. Based on the previous results on BNYVV 14K protein and the functional prediction on RBSDV S6 protein by com-puter analysis, in which both proteins showed activities to bind with RNAs, they may bind with specific siRNAs in RITS and affect the gene methylations.

Since the plant symptoms caused by PVX infection were aggravated by immixture of BNYVV 14K or RBSDV S6 genes (Fig. 5), which is similar to those on N. benthamiana by PVY HC-Pro or CMV 2b[26], it is specu-lated that these two genes may have the suppressing func-tion. In trans-changes experiment of gene silencing as previous report[27], pDN14K and pNDS6 were used for the second infiltration of the silenced N. benthamiana-16C respectively, in which the silencing signal transduction was partially blocked into the apical meristems at 30 dpi, as well as that induced by CMV 2b gene.

The study on virus-encoded suppressor will be help-ful for our understanding of the mechanism of gene si-lencing. However, since the interaction of gene silencing and suppressing is very complex, different pathways[28] and various proteins[29] were involved and hardly separa-ble from each other. Because of the differences in the vectors used to carry the target gene, the constructions of expression cassette in the vector and the growth stages of plants to be inoculated, distinguished results may be ob-tained from analysis of the same virus-encoded suppressor. Future investigations to elucidate these questions will fo-cus on the vector construction and expression dosage that affects gene silencing. Acknowladgements The authors thank Dr. D.C. Baulcombe at Sains-bury laboratory, John Innes Center, UK for kindly providing the materi-als. This work was supported by the National Natural Science Founda-tion of China (Grants Nos. 30325001 and 30471136).

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(Received December 5, 2004; accepted January 5, 2005)