- Email: [email protected]
Construction of an infectious cDNA clone and gene expression vector of Tobacco vein banding mosaic virus (genus Potyvirus)
Virus Research 169 (2012) 276–281
Contents lists available at SciVerse ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Construction of an infectious cDNA clone and gene expression vector of Tobacco vein banding mosaic virus (genus Potyvirus) Rui Gao a,b,c,1 , Yan-Ping Tian b,1 , Jie Wang a,1 , Xiao Yin a , Xiang-Dong Li a,∗ , Jari P.T. Valkonen b a b c
Laboratory of Plant Virology, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an, Shandong 271018, China Department of Agricultural Sciences, PO Box 27, FI-00014 University of Helsinki, Finland Department of Microbiology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 19 April 2012 Received in revised form 12 July 2012 Accepted 12 July 2012 Available online 20 July 2012 Keywords: TVBMV Plant virus Potyvirus Infectious clone Gene vector Tobacco
a b s t r a c t Tobacco vein banding mosaic virus (TVBMV, genus Potyvirus) mainly infects solanaceous plants and is of increasing economic importance in China. Here, we report sequence determination of the full-length 5 untranslated region of TVBMV isolate HN39 and construction of an infectious clone. The resultant clone, pTVBMV, which was stabilized by introducing three introns in the P3 and CI-encoding regions, induced similar disease symptoms and accumulated similar titers of virus in plants of Nicotiana benthamiana, Nicotiana tabacum and N. rustica as the wild type HN39 isolate. Mutation of arginine to isoleucine (R182I) or aspartic acid to lysine (D198K) in HC-Pro alleviated the symptoms of pTVBMV significantly, indicating a role of the two amino acids in regulating virulence of TVBMV. The Aequoria victoriae gene for green fluorescent protein was inserted between the NIb and CP encoding regions of pTVBMV and expressed stably in the systemically infected N. benthamiana leaves, indicating suitability of pTVBMV for expression of foreign proteins in plants. © 2012 Elsevier B.V. All rights reserved.
Construction of full-length infectious cDNA clones is critical for the functional analysis of RNA viruses. Infectious cDNA clones will allow mapping the viral determinants involved in virus replication, local and systemic movement and symptom development, and interactions between viral and host factors during virus infection in plants (Nagyová and Subr, 2007). Infectious cDNA clones of viruses can also be developed into vectors for foreign gene expression or virus induced gene silencing (Holzberg et al., 2002; Kelloniemi et al., 2008; Lacomme et al., 2003; Lindbo, 2007; Ratcliff et al., 2001; Sainsbury et al., 2010). There are two major strategies for constructing infectious cDNA clones of plant viruses. The first one is to place the viral cDNA under the promoter SP6, T3 or T7 to produce infectious viral RNA via in vitro transcription (Nagyová and Subr, 2007). These promoters are small and can be synthesized as part of the primers used for cloning. However, the costs of use of infectious viral clones made by this method are high because viral RNA needs to be synthesized and, in some viruses, viral RNAs need to be capped before use for inoculation. The second strategy is to use the Cauliflower mosaic virus (CaMV) 35S promoter, by which infectious RNA can be produced via in vivo transcription. This latter method is less expensive
∗ Corresponding author. Tel.: +86 538 8242523; fax: +86 538 8226399. E-mail addresses: [email protected], [email protected] (X.-D. Li). 1 These authors contributed equally. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.07.010
and more practical, but may suffer from low infectivity levels when mechanical rather than biolistic inoculation is carried out (Jakab et al., 1997; Nagyová and Subr, 2007). The viral genome may contain promoter-like elements resulting in production of toxic products in Escherichia coli cells during cloning, and spontaneous mutations, deletions and rearrangements may occur during propagation of the plasmids in E. coli cells (Jakab et al., 1997; Johansen, 1996; Johansen and Lund, 2008). To alleviate such problems, AU-rich plant introns can be inserted into viral genomes to terminate expression of undesired toxic proteins in E. coli cells. Following introduction of the transcripts into plant cells, the intron sequences are removed precisely to produce the full length infectious RNA of the virus (González et al., 2002; Johansen, 1996; Johansen and Lund, 2008; López-Moya and García, 2000; Olsen and Johansen, 2001; Ülper et al., 2008; Yang et al., 1998). However, the number and/or locations of intron insertions needed to stabilize individual infectious clones vary, even for clones representing closely related viruses (Johansen, 1996). Tobacco vein banding mosaic virus (TVBMV) is a potyvirus of increasing economic importance in China due to the yield losses it causes in tobacco production (Chen et al., 2009; Tian et al., 2007). Like other potyviruses, the TVBMV genome contains a single large open reading frame (ORF) that encodes a polyprotein, which is cleaved into ten mature proteins by three self-encoded proteinases. Additionally, a small protein is translated from an ORF residing within the P3-encoding sequences by a frame-shifting strategy
R. Gao et al. / Virus Research 169 (2012) 276–281
277
Fig. 1. Construction of the infectious clone pTVBMV. wtTVBMV shows the genome organization of TVBMV. p35S-HC2110 , pHC2111 -6K26075 and p6K26076 -PolyA are the three intermediate plasmids used to construct the full length cDNA clone of TVBMV. pTVBMV shows the structure of the infectious clone of TVBMV and the positions of three introns inserted to stabilize the clone. The mature viral proteins: P1, the first protein; HC-Pro, helper component proteinase; P3, third protein; 6K1 and 6K2, 6 kDa proteins; CI, cylindrical inclusion protein; VPg, viral genome-linked protein; NIa-Pro, the main viral proteinase; NIb, replicase; and CP, coat protein. A(18) indicates the poly(A) tail. The 5 and 3 untranslated regions (UTR) are depicted.
(Chung et al., 2008; Zhang et al., 2011). TVBMV isolates are clustered phylogenetically to three groups which correlate with the geographic origin (Tian et al., 2007). The genome sequences of TVBMV isolates HN39 and YND, which differ in virulence and belong to different strains, have been determined (Wang et al., 2010; Yu et al., 2007). Selection, gene flow and recombination are important factors driving the evolution of TVBMV in nature (Zhang et al., 2011). In a previous study, presence of only box ‘b’ was revealed in the 5 -untranslated region (5 -UTR) of TVBMV-HN39 genome (NCBI sequence accession number EU734432; Wang et al., 2010). In the present study, the 5 -UTR of HN39 was determined using the 5 -RACE method (Scotto-Lavino et al., 2007). HN39 was maintained in Nicotiana tabacum cv. Samsun plants grown in a growth chamber under 16 h photoperiod (110 E m−2 s−1 ; illumination of fluorescent lamps alternately with tubes of 58 W/830 and 36 W/77) at 23 ◦ C under 40% relative humidity. Plants were watered when needed and fertilized weekly with 1% N:P:K = 16:9:22 fertilizer (Yara, Espoo, Finland). TVBMV particles were purified from infected Samsun leaves as previously reported (Fribourg and Nakashima, 1984). Viral RNA was extracted from purified particles as described by Monger et al. (2001). The amplification products were gel-purified and ligated into the pGEM-T vector, followed by transformation into the DH5˛ competent cells. Plasmids were isolated from individual colonies using GenElute plasmid purification kit as instructed (Sigma–Aldrich, Steinheim, Germany). Two independent 5 -RACE reactions were made, and ten clones were sequenced to determine the first nucleotides of the TVBMV HN39 5 -UTR. The length of 5 -UTR varied in different clones. Among the ten clones sequenced, three had the longest 5 -UTR of 171 nucleotides (nt; accession number JQ407081), which is 25 nt longer than that reported previously and contained both boxes ‘a’ (ACAACAU) and ‘b’ (UCAAGCA) sequences that are highly conserved in the Potyviridae family (Simón-Buela et al., 1997). To construct a full-length cDNA clone of TVBMV HN39, three fragments covering the full length genomic sequence were prepared (Fig. 1 and Suppl. Table 1). p35S-HC2110 contained the 35S promoter and the first 2110 nt of the 5 -part of TVBMV genome. pHC2111 -6K26075 contained TVBMV nt 2111–6075 and three introns. p6K26076 -SmaI covered the 3 -part of TVMBV genome from nt 6076 to the 3 -end poly(A) tail and contained a SmaI restriction site at the end. The three fragments were used to assemble the full-length TVBMV clone pTVBMV. To stabilize the clone, three introns (one in the P3-encoding region and two in the CI-encoding region) amplified from the infectious clone for Potato virus Y (PVY) (Bukovinszki et al., 2007) were inserted at nt positions 3141, 3981 and 5380, respectively (Fig. 1 and Suppl. Table 1). Removal of one or two introns from pTVBMV resulted in deletions or rearrangements of the viral sequence, as tested by restriction analysis of the
plasmid, which further emphasized the importance of the introns in stabilizing the viral genome in the plasmid. To test infectivity, pTVBMV was inoculated to fully developed leaves of 6-week-old Nicotiana benthamiana plants by particle bombardment (Sikorskaite et al., 2010). Nine plants were inoculated with each plasmid and experiments were repeated for three times. The inoculated plants were grown in growth chambers at 22 ◦ C under a 16-h photoperiod (relative humidity 75%). Leaf discs from systemically infected leaves of four inoculated plants for each treatment were collected at 5, 8, 11, 14, 17 and 20 days post-inoculation (dpi). Virus accumulation levels were determined by indirect platetrapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using TVBMV coat protein (CP) specific antibodies (Lan et al., 2007). Absorbance values (A405 ) were recorded for each sample using a Benchmark plate reader (Bio-Rad). Total RNA was extracted from infected leaves using a homemade TRIzol as described (Caldo et al., 2004). First-strand cDNA synthesis was done using an oligo(dT) primer and M-MLV reverse transcriptase (Promega). Viral RNA accumulation in leaves was monitored by quantitative real time reverse transcription PCR (qRT-PCR) using specific primers TVBMV CP-F and TVBMV CP-R (Suppl. Table 1). Systemically infected leaves in all plants inoculated with pTVBMV showed vein clearing symptoms similar to the original TVBMV-HN39 by 8 dpi (Fig. 2A), followed by development of severe mosaic symptoms in the youngest leaves and stunting of the plants by 14 dpi. Sequencing of progeny viruses from the pTVBMVinoculated plants showed that all three introns had been removed completely and precisely. Crude extracts were prepared from the leaves of N. benthamiana systemically infected with either the pTVBMV-derived virus or wild type HN39 and used for mechanical inoculation of seedlings of N. benthamiana, N. tabacum and N. rustica. By 5 dpi all the inoculated plants showed similar vein clearing and mosaic symptoms in the systemically infected leaves, indicating that the progeny viruses were spreading systemically in these host plants at the same rate. Analysis of the plants by PTA-ELISA showed that both viruses accumulated to similar levels in the host plants (Fig. 2B). To test importance of the conserved motifs FR182 NK and CD198 N in HC-Pro on virulence of TVBMV, substitutions of isoleucine for arginine (R182I) and lysine for aspartic acid (D198K) were introduced to modify the motifs, respectively, using primers (Suppl. Table 1) designed as described by Liu and Naismith (2008). The mutated clones were verified by sequencing and no additional mutations were detected. Inoculation of N. benthamiana plants showed that the pTVBMV-derived virus caused mosaic and distortion in the systemically infected leaves and stunting of the plants as before. In contrast, the viruses derived from pTVBMVR182I and pTVBMVD198K carrying a mutation in the motifs FRNK and CDN,
278
R. Gao et al. / Virus Research 169 (2012) 276–281
Fig. 2. Symptoms and accumulation levels of the virus derived from pTVBMV in three test plant species. (A) Symptoms induced by wild-type TVBMV strain HN39 (wtTVBMV) and the virus derived from pTVBMV in the upper non-inoculated leaves 8 days post-inoculation. In Nicotiana benthamiana, both viruses induce mild vein clearing and epinasty in the expanding young leaves. In N. tabacum cv. Samsun, only mild vein clearing is observed. In N. rustica plants, vein clearing is apparent in the basal part of the leaf at 8 days post-inoculation, whereas the tip of the illustrated leaves shows no symptoms. (B) Virus accumulation in the systemically infected leaves as determined by PTA-ELISA at 8 days post-inoculation.
respectively, caused only mild vein clearing if any symptoms in the systemically infected leave (Fig. 3A). Virus accumulation in the systemically infected leaves of N. benthamiana plants inoculated with pTVBMVR182I or pTVBMVD198K was too low to be detected by PTAELISA (Fig. 3B), but TVBMV RNA was detected by qRT-PCR analysis using TVBMV specific primers (Fig. 3C). Our results indicate that mutations R182I and D198K in HC-Pro of TVBMV have a significant impact on virus accumulation and symptom expression in the systemic leaves of N. benthamiana. In order to evaluate the possibility of using TVBMV as an expression vector, the green fluorescent protein gene gfp of Aequoria victoriae was inserted into pTVBMV between the NIband CP-encoding regions to generate the GFP-expressing plasmid pTVBMV-GFP by overlapping PCR (Fig. 4A) (Chalfie et al., 1994). The primers used to construct the plasmid pTVBMV-GFP are listed in Suppl. Table 1. The pTVBMV-GFP vector was inoculated to N. benthamiana by particle bombardment and systemic movement
was detected in the upper leaves of the inoculated plants by GFP expression under UV-light. At 8 dpi, the plants inoculated with pTVBMV-GFP showed vein clearing symptoms similar to those of TVBMV-HN39 in the systemically infected leaves (Fig. 4B), indicating that GFP insertion did not affect virus infection and movement significantly. Green fluorescence was observed in and around the mid-vein and minor veins of the systemically infected leaves. By 14 dpi, these leaves showed strong green fluorescence in the systemically infected leaves, implying that GFP can be expressed stably to a high level (Fig. 4B). Virus species in the genus Potyvirus (family Potyviridae) are divided to several phylogenetic subgroups based on phylogenetic analyses (Adams et al., 2012). TVBMV is placed in the same group with Chronic bee paralysis virus, Wild tomato mosaic virus, Chilli veinal mottle virus and Pepper veinal mottle virus. To our knowledge, TVBMV is the first virus of this subgroup for which an infectious cDNA is now available. The P3-encoding sequence in several
R. Gao et al. / Virus Research 169 (2012) 276–281
279
Fig. 3. Symptoms and virus accumulation in N. benthamiana plants inoculated with pTVBMV and two mutants pTVBMVR182I and pTVBMVD198K . (A) Following inoculation with pTVBMV, the systemically infected leaf displays apparent vein clearing symptoms, whereas mild or no symptoms are observed in the leaves of plants inoculated with pTVBMVR182I and pTVBMVD198K 8 days post-inoculation. (B) Accumulation of TVBMV in the upper non-inoculated leaves of N. benthamiana plants at various times post inoculation, as determined by PTA-ELISA. (C) Detection of TVBMV RNA in the systemically infected leaves by qRT-PCR at 11 days post-inoculation. Levels of actin transcripts in these tissues were used as an internal control.
potyviruses is claimed to contain cryptic promoters that are active in bacterial cells and drive expression of proteins toxic to bacteria (Subr et al., 2000). This problem can be solved by insertion of intron(s) in the viral clones (Johansen, 1996; Johansen and Lund,
2008; López-Moya and García, 2000; Olsen and Johansen, 2001; Yamshchikov et al., 2001). For example, insertion of an intron within the P3-encoding region stabilized the infectious clone of Plum pox virus (López-Moya and García, 2000), and insertion of
Fig. 4. Expression of GFP using the vector pTVBMV-GFP GFP fluorescence was observed under UV light in the N. benthamiana leaves systemically infected with pTVBMV-GFP at 8 and 14 days post-inoculation (dpi).
280
R. Gao et al. / Virus Research 169 (2012) 276–281
introns into the P1, P3, CI and 6K2 encoding regions stabilized the full length clone of Pea seed-borne mosaic virus in E. coli (Olsen and Johansen, 2001). In our study, insertion of an intron into the P3-encoding sequence alone was not sufficient to stabilize the full length clone of TVBMV, but two additional introns in the CIencoding sequence stabilized the viral cDNA. Similar results have been obtained when introns were inserted in the P3- and CIencoding regions of the clone PVY-N605(123) (Bukovinszki et al., 2007). Site-directed mutagenesis of the infectious clone showed that the FRNK and CDN motifs of HC-Pro are involved in virulence of TVBMV. When the lysine was substituted for aspartic acid in the CDN motif of TVBMV HC-Pro, the virus became less virulent and the mutant virus accumulated to lower levels in infected plants than the wild type virus. The CDN motif has been reported to play a role in virus accumulation and symptom development also in other potyviruses (Desbiez et al., 2010; Gal-On, 2000; Hu et al., 2009; Torres-Barceló et al., 2008; Yambao et al., 2008). Mutation of the HC-Pro FRNK motif to FINK reduced both virulence and accumulation of TVBMV in plants, whereas a similar mutation in HC-Pro in Zucchini yellow mosaic virus has no such effect (Shiboleth et al., 2007). We also demonstrated with the construct pTVBMV-GFP that TVBMV has potential in use as a gene expression vector in plants. Stability of the recombinant viruses containing foreign genes often appears as a bottleneck for the applications (Chung et al., 2007), but our data indicate that pTVMBV-GFP is stable. Taken together, we have successfully constructed an infectious cDNA clone of TVBMV, which can be easily and cost-effectively delivered into plants with a portable biolistic device (HandyGun) that can be prepared from commonly available parts (Sikorskaite et al., 2010). The infectious TVBMV clone enables us to study virulence of TVBMV in plants, engineer mild strains for use in crossprotection against severe strains of TVBMV, and develop vectors for expression of foreign proteins in plants. Acknowledgments This study was supported by Special Research Funds for Doctoral Program (SRFDP, 20080434006; 20123702110013) from Ministry of Education, China, grants from National Natural Science Foundation of China (NSFC; 30971895 and 31011130031) and the Academy of Finland (1134759 and 1253126). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2012.07.010. References Adams, M.J., Zerbini, F.M., Stenger, D.C., Rabenstein, D., French, R., Valkonen, J.P.T., 2012. Family – Potyviridae. In: Andrew, M.Q.K., Elliot, L., Michael, J.A., Carstens, E.B. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses: The Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, pp. 1069–1089. Bukovinszki, Á., Götz, R., Johansen, E., Maiss, E., Balázs, E., 2007. The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains. Virus Research 127, 122–125. Caldo, R.A., Nettleton, D., Wise, R.P., 2004. Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16, 2514–2528. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802–805. Chen, X.-Z., Wen, L., Wei, D., Gao, R., Yuan, C.Y., Li, X.-D., 2009. Detection of viruses infecting tobacco in Mengyin. Shandong Agricultural Sciences 10, 62–63. Chung, B.N., Canto, T., Palukaitis, P., 2007. Stability of recombinant plant viruses containing genes of unrelated plant viruses. Journal of General Virology 88, 1347–1355.
Chung, B.Y.W., Miller, W.A., Atkins, J.F., Firth, A.E., 2008. An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences of the United States of America 105, 5897–5902. Desbiez, C., Girard, M., Lecoq, H., 2010. A novel natural mutation in HC-Pro responsible for mild symptomatology of Zucchini yellow mosaic virus (ZYMV, Potyvirus) in cucurbits. Archives of Virology 155, 397–401. Fribourg, C.E., Nakashima, J., 1984. Characterization of a new potyvirus from potato. Phytopathology 74, 1363–1369. Gal-On, A., 2000. A point mutation in the FRNK motif of the Potyvirus Helper Component-protease gene alters symptom expression in cucurbits and elicits protection against the severe homologous virus. Phytopathology 90, 467–473. González, J.M., Pénzes, Z., Almazán, F., Calvo, E., Enjuanes, L., 2002. Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. Journal of Virology 76, 4655–4661. Holzberg, S., Brosio, P., Gross, C., Pogue, G.P., 2002. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant Journal 30, 315–327. Hu, X., Meacham, T., Ewing, L., Gray, S.M., Karasev, A.V., 2009. A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco. Virus Research 143, 68–76. Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. The Journal of General Virology 78, 3141–3145. Johansen, I.E., 1996. Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo. Proceedings of the National Academy of Sciences of the United States of America 93, 12400–12405. Johansen, I.E., Lund, O., 2008. Insertion of introns: a strategy to facilitate assembly of infectious full length clones. In: Foster, G., Johansen, I.E., Hong, Y., Nagy, P. (Eds.), Plant Virology Protocols, vol. 451. Humana Press, pp. 535–544. Kelloniemi, J., Mäkinen, K., Valkonen, J.P.T., 2008. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Research 135, 282–291. Lacomme, C., Hrubikova, K., Hein, I., 2003. Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. The Plant Journal 34, 543–553. Lan, Y.-F., Liu, J.-L., Gao, R., Wang, H.-Y., Zhu, T.-S., Zhu, X.-P., Li, X.-D., 2007. Expression of Tobacco vein banding mosaic virus coat protein in E. coli and preparation of antiserum. Acta Phytopathologica Sinica 37, 461–466 (in Chinese). Lindbo, J.A., 2007. TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiology 145, 1232–1240. Liu, H., Naismith, J.H., 2008. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology 8, 91. López-Moya, J.J., García, J.A., 2000. Construction of a stable and highly infectious intron-containing cDNA clone of Plum pox potyvirus and its use to infect plants by particle bombardment. Virus Research 68, 99–107. Monger, W.A., Seal, S., Isaac, A.M., Foster, G.D., 2001. Molecular characterization of the Cassava brown streak virus coat protein. Plant Pathology 50, 527–534. Nagyová, A., Subr, Z., 2007. Infectious full-length clones of plant viruses and their use for construction of viral vectors. Acta Virologica 51, 223–237. Olsen, B.S., Johansen, I.E., 2001. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus. Archives of Virology 146, 15–25. Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C., 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant Journal 25, 237–245. Sainsbury, F., Canizares, M.C., Lomonossoff, G.P., 2010. Cowpea mosaic virus: the plant virus-based biotechnology workhorse. Annual Review of Phytopathology 48, 437–455. Scotto-Lavino, E., Du, G., Frohman, M.A., 2007. 5 end cDNA amplification using classic RACE. Nature Protocols 1, 2555–2562. Shiboleth, Y.M., Haronsky, E., Leibman, D., Arazi, T., Wassenegger, M., Whitham, S.A., Gaba, V., Gal-On, A., 2007. The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. Journal of Virology 81, 13135–13148. Sikorskaite, S., Vuorinen, A.L., Rajamäki, M.-L., Nieminen, A., Gaba, V., Valkonen, J.P.T., 2010. HandyGun: an improved custom-designed, non-vacuum gene gun suitable for virus inoculation. Journal of Virological Methods 165, 320–324. Simón-Buela, L., Guo, H.S., García, J.A., 1997. Long sequences in the 5 noncoding region of Plum pox virus are not necessary for viral infectivity but contribute to viral competitiveness and pathogenesis. Virology 233, 157–162. Subr, Z., Fomitcheva, V.W., Ühne, T.K., 2000. The complete nucleotide sequence of RNA1 of Barley mild mosaic virus (ASL1) and attempts at bacterial expression of the P3 protein. Journal of Phytopathology 148, 461–467. Tian, Y.P., Liu, J.L., Yu, X.Q., Lei, L.P., Zhu, X.P., Valkonen, J.P.T., Li, X.D., 2007. Molecular diversity of Tobacco vein banding mosaic virus. Archives of Virology 152, 1911–1915. Torres-Barceló, C., Martín, S., Daròs, J.-A., Elena, S.F., 2008. From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro. Genetics 180, 1039–1049. Ülper, L., Sarand, I., Rausalu, K., Merits, A., 2008. Construction, properties, and potential application of infectious plasmids containing Semliki Forest virus full-length cDNA with an inserted intron. Journal of Virological Methods 148, 265–270. Wang, H.Y., Zhu, T.S., Cui, T.T., Hou, S.S., Yin, X., Li, X.-D., Lei, L.P., Zhu, X.P., 2010. Complete genome sequence of a tobacco isolate of the Tobacco vein banding mosaic virus strain prevailing in China. Archives of Virology 155, 293–295. Yambao, M.L.M., Yagihashi, H., Sekiguchi, H., Sekiguchi, T., Sasaki, T., Sato, M., Atsumi, G., Tacahashi, Y., Nakahara, K.S., Uyeda, I., 2008. Point mutations in helper
R. Gao et al. / Virus Research 169 (2012) 276–281 component protease of Clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean. Archives of Virology 153, 105–115. Yamshchikov, V., Mishin, V., Cominelli, F., 2001. A new strategy in design of (+)RNA virus infectious clones enabling their stable propagation in E. coli. Virology 281, 272–280. Yang, S.J., Revers, F., Souche, S., Lot, H., Le Gall, O., Candresse, T., Dunez, J., 1998. Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects
281
of intron-insertion on their viability in Escherichia coli and on their infectivity to plants. Archives of Virology 143, 2443–2451. Yu, X.-Q., Lan, Y.-F., Wang, H.-Y., Liu, J.-L., Zhu, X.-P., Valkonen, J.P.T., Li, X.-D., 2007. The complete genomic sequence of Tobacco vein banding mosaic virus and its similarities with other potyviruses. Virus Genes 35, 801–806. Zhang, C.-L., Gao, R., Wang, J., Zhang, G.-M., Li, X.-D., Liu, H.-T., 2011. Molecular variability of Tobacco vein banding mosaic virus populations. Virus Research 158, 188–198.
Contents lists available at SciVerse ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Construction of an infectious cDNA clone and gene expression vector of Tobacco vein banding mosaic virus (genus Potyvirus) Rui Gao a,b,c,1 , Yan-Ping Tian b,1 , Jie Wang a,1 , Xiao Yin a , Xiang-Dong Li a,∗ , Jari P.T. Valkonen b a b c
Laboratory of Plant Virology, Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an, Shandong 271018, China Department of Agricultural Sciences, PO Box 27, FI-00014 University of Helsinki, Finland Department of Microbiology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 19 April 2012 Received in revised form 12 July 2012 Accepted 12 July 2012 Available online 20 July 2012 Keywords: TVBMV Plant virus Potyvirus Infectious clone Gene vector Tobacco
a b s t r a c t Tobacco vein banding mosaic virus (TVBMV, genus Potyvirus) mainly infects solanaceous plants and is of increasing economic importance in China. Here, we report sequence determination of the full-length 5 untranslated region of TVBMV isolate HN39 and construction of an infectious clone. The resultant clone, pTVBMV, which was stabilized by introducing three introns in the P3 and CI-encoding regions, induced similar disease symptoms and accumulated similar titers of virus in plants of Nicotiana benthamiana, Nicotiana tabacum and N. rustica as the wild type HN39 isolate. Mutation of arginine to isoleucine (R182I) or aspartic acid to lysine (D198K) in HC-Pro alleviated the symptoms of pTVBMV significantly, indicating a role of the two amino acids in regulating virulence of TVBMV. The Aequoria victoriae gene for green fluorescent protein was inserted between the NIb and CP encoding regions of pTVBMV and expressed stably in the systemically infected N. benthamiana leaves, indicating suitability of pTVBMV for expression of foreign proteins in plants. © 2012 Elsevier B.V. All rights reserved.
Construction of full-length infectious cDNA clones is critical for the functional analysis of RNA viruses. Infectious cDNA clones will allow mapping the viral determinants involved in virus replication, local and systemic movement and symptom development, and interactions between viral and host factors during virus infection in plants (Nagyová and Subr, 2007). Infectious cDNA clones of viruses can also be developed into vectors for foreign gene expression or virus induced gene silencing (Holzberg et al., 2002; Kelloniemi et al., 2008; Lacomme et al., 2003; Lindbo, 2007; Ratcliff et al., 2001; Sainsbury et al., 2010). There are two major strategies for constructing infectious cDNA clones of plant viruses. The first one is to place the viral cDNA under the promoter SP6, T3 or T7 to produce infectious viral RNA via in vitro transcription (Nagyová and Subr, 2007). These promoters are small and can be synthesized as part of the primers used for cloning. However, the costs of use of infectious viral clones made by this method are high because viral RNA needs to be synthesized and, in some viruses, viral RNAs need to be capped before use for inoculation. The second strategy is to use the Cauliflower mosaic virus (CaMV) 35S promoter, by which infectious RNA can be produced via in vivo transcription. This latter method is less expensive
∗ Corresponding author. Tel.: +86 538 8242523; fax: +86 538 8226399. E-mail addresses: [email protected], [email protected] (X.-D. Li). 1 These authors contributed equally. 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.07.010
and more practical, but may suffer from low infectivity levels when mechanical rather than biolistic inoculation is carried out (Jakab et al., 1997; Nagyová and Subr, 2007). The viral genome may contain promoter-like elements resulting in production of toxic products in Escherichia coli cells during cloning, and spontaneous mutations, deletions and rearrangements may occur during propagation of the plasmids in E. coli cells (Jakab et al., 1997; Johansen, 1996; Johansen and Lund, 2008). To alleviate such problems, AU-rich plant introns can be inserted into viral genomes to terminate expression of undesired toxic proteins in E. coli cells. Following introduction of the transcripts into plant cells, the intron sequences are removed precisely to produce the full length infectious RNA of the virus (González et al., 2002; Johansen, 1996; Johansen and Lund, 2008; López-Moya and García, 2000; Olsen and Johansen, 2001; Ülper et al., 2008; Yang et al., 1998). However, the number and/or locations of intron insertions needed to stabilize individual infectious clones vary, even for clones representing closely related viruses (Johansen, 1996). Tobacco vein banding mosaic virus (TVBMV) is a potyvirus of increasing economic importance in China due to the yield losses it causes in tobacco production (Chen et al., 2009; Tian et al., 2007). Like other potyviruses, the TVBMV genome contains a single large open reading frame (ORF) that encodes a polyprotein, which is cleaved into ten mature proteins by three self-encoded proteinases. Additionally, a small protein is translated from an ORF residing within the P3-encoding sequences by a frame-shifting strategy
R. Gao et al. / Virus Research 169 (2012) 276–281
277
Fig. 1. Construction of the infectious clone pTVBMV. wtTVBMV shows the genome organization of TVBMV. p35S-HC2110 , pHC2111 -6K26075 and p6K26076 -PolyA are the three intermediate plasmids used to construct the full length cDNA clone of TVBMV. pTVBMV shows the structure of the infectious clone of TVBMV and the positions of three introns inserted to stabilize the clone. The mature viral proteins: P1, the first protein; HC-Pro, helper component proteinase; P3, third protein; 6K1 and 6K2, 6 kDa proteins; CI, cylindrical inclusion protein; VPg, viral genome-linked protein; NIa-Pro, the main viral proteinase; NIb, replicase; and CP, coat protein. A(18) indicates the poly(A) tail. The 5 and 3 untranslated regions (UTR) are depicted.
(Chung et al., 2008; Zhang et al., 2011). TVBMV isolates are clustered phylogenetically to three groups which correlate with the geographic origin (Tian et al., 2007). The genome sequences of TVBMV isolates HN39 and YND, which differ in virulence and belong to different strains, have been determined (Wang et al., 2010; Yu et al., 2007). Selection, gene flow and recombination are important factors driving the evolution of TVBMV in nature (Zhang et al., 2011). In a previous study, presence of only box ‘b’ was revealed in the 5 -untranslated region (5 -UTR) of TVBMV-HN39 genome (NCBI sequence accession number EU734432; Wang et al., 2010). In the present study, the 5 -UTR of HN39 was determined using the 5 -RACE method (Scotto-Lavino et al., 2007). HN39 was maintained in Nicotiana tabacum cv. Samsun plants grown in a growth chamber under 16 h photoperiod (110 E m−2 s−1 ; illumination of fluorescent lamps alternately with tubes of 58 W/830 and 36 W/77) at 23 ◦ C under 40% relative humidity. Plants were watered when needed and fertilized weekly with 1% N:P:K = 16:9:22 fertilizer (Yara, Espoo, Finland). TVBMV particles were purified from infected Samsun leaves as previously reported (Fribourg and Nakashima, 1984). Viral RNA was extracted from purified particles as described by Monger et al. (2001). The amplification products were gel-purified and ligated into the pGEM-T vector, followed by transformation into the DH5˛ competent cells. Plasmids were isolated from individual colonies using GenElute plasmid purification kit as instructed (Sigma–Aldrich, Steinheim, Germany). Two independent 5 -RACE reactions were made, and ten clones were sequenced to determine the first nucleotides of the TVBMV HN39 5 -UTR. The length of 5 -UTR varied in different clones. Among the ten clones sequenced, three had the longest 5 -UTR of 171 nucleotides (nt; accession number JQ407081), which is 25 nt longer than that reported previously and contained both boxes ‘a’ (ACAACAU) and ‘b’ (UCAAGCA) sequences that are highly conserved in the Potyviridae family (Simón-Buela et al., 1997). To construct a full-length cDNA clone of TVBMV HN39, three fragments covering the full length genomic sequence were prepared (Fig. 1 and Suppl. Table 1). p35S-HC2110 contained the 35S promoter and the first 2110 nt of the 5 -part of TVBMV genome. pHC2111 -6K26075 contained TVBMV nt 2111–6075 and three introns. p6K26076 -SmaI covered the 3 -part of TVMBV genome from nt 6076 to the 3 -end poly(A) tail and contained a SmaI restriction site at the end. The three fragments were used to assemble the full-length TVBMV clone pTVBMV. To stabilize the clone, three introns (one in the P3-encoding region and two in the CI-encoding region) amplified from the infectious clone for Potato virus Y (PVY) (Bukovinszki et al., 2007) were inserted at nt positions 3141, 3981 and 5380, respectively (Fig. 1 and Suppl. Table 1). Removal of one or two introns from pTVBMV resulted in deletions or rearrangements of the viral sequence, as tested by restriction analysis of the
plasmid, which further emphasized the importance of the introns in stabilizing the viral genome in the plasmid. To test infectivity, pTVBMV was inoculated to fully developed leaves of 6-week-old Nicotiana benthamiana plants by particle bombardment (Sikorskaite et al., 2010). Nine plants were inoculated with each plasmid and experiments were repeated for three times. The inoculated plants were grown in growth chambers at 22 ◦ C under a 16-h photoperiod (relative humidity 75%). Leaf discs from systemically infected leaves of four inoculated plants for each treatment were collected at 5, 8, 11, 14, 17 and 20 days post-inoculation (dpi). Virus accumulation levels were determined by indirect platetrapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using TVBMV coat protein (CP) specific antibodies (Lan et al., 2007). Absorbance values (A405 ) were recorded for each sample using a Benchmark plate reader (Bio-Rad). Total RNA was extracted from infected leaves using a homemade TRIzol as described (Caldo et al., 2004). First-strand cDNA synthesis was done using an oligo(dT) primer and M-MLV reverse transcriptase (Promega). Viral RNA accumulation in leaves was monitored by quantitative real time reverse transcription PCR (qRT-PCR) using specific primers TVBMV CP-F and TVBMV CP-R (Suppl. Table 1). Systemically infected leaves in all plants inoculated with pTVBMV showed vein clearing symptoms similar to the original TVBMV-HN39 by 8 dpi (Fig. 2A), followed by development of severe mosaic symptoms in the youngest leaves and stunting of the plants by 14 dpi. Sequencing of progeny viruses from the pTVBMVinoculated plants showed that all three introns had been removed completely and precisely. Crude extracts were prepared from the leaves of N. benthamiana systemically infected with either the pTVBMV-derived virus or wild type HN39 and used for mechanical inoculation of seedlings of N. benthamiana, N. tabacum and N. rustica. By 5 dpi all the inoculated plants showed similar vein clearing and mosaic symptoms in the systemically infected leaves, indicating that the progeny viruses were spreading systemically in these host plants at the same rate. Analysis of the plants by PTA-ELISA showed that both viruses accumulated to similar levels in the host plants (Fig. 2B). To test importance of the conserved motifs FR182 NK and CD198 N in HC-Pro on virulence of TVBMV, substitutions of isoleucine for arginine (R182I) and lysine for aspartic acid (D198K) were introduced to modify the motifs, respectively, using primers (Suppl. Table 1) designed as described by Liu and Naismith (2008). The mutated clones were verified by sequencing and no additional mutations were detected. Inoculation of N. benthamiana plants showed that the pTVBMV-derived virus caused mosaic and distortion in the systemically infected leaves and stunting of the plants as before. In contrast, the viruses derived from pTVBMVR182I and pTVBMVD198K carrying a mutation in the motifs FRNK and CDN,
278
R. Gao et al. / Virus Research 169 (2012) 276–281
Fig. 2. Symptoms and accumulation levels of the virus derived from pTVBMV in three test plant species. (A) Symptoms induced by wild-type TVBMV strain HN39 (wtTVBMV) and the virus derived from pTVBMV in the upper non-inoculated leaves 8 days post-inoculation. In Nicotiana benthamiana, both viruses induce mild vein clearing and epinasty in the expanding young leaves. In N. tabacum cv. Samsun, only mild vein clearing is observed. In N. rustica plants, vein clearing is apparent in the basal part of the leaf at 8 days post-inoculation, whereas the tip of the illustrated leaves shows no symptoms. (B) Virus accumulation in the systemically infected leaves as determined by PTA-ELISA at 8 days post-inoculation.
respectively, caused only mild vein clearing if any symptoms in the systemically infected leave (Fig. 3A). Virus accumulation in the systemically infected leaves of N. benthamiana plants inoculated with pTVBMVR182I or pTVBMVD198K was too low to be detected by PTAELISA (Fig. 3B), but TVBMV RNA was detected by qRT-PCR analysis using TVBMV specific primers (Fig. 3C). Our results indicate that mutations R182I and D198K in HC-Pro of TVBMV have a significant impact on virus accumulation and symptom expression in the systemic leaves of N. benthamiana. In order to evaluate the possibility of using TVBMV as an expression vector, the green fluorescent protein gene gfp of Aequoria victoriae was inserted into pTVBMV between the NIband CP-encoding regions to generate the GFP-expressing plasmid pTVBMV-GFP by overlapping PCR (Fig. 4A) (Chalfie et al., 1994). The primers used to construct the plasmid pTVBMV-GFP are listed in Suppl. Table 1. The pTVBMV-GFP vector was inoculated to N. benthamiana by particle bombardment and systemic movement
was detected in the upper leaves of the inoculated plants by GFP expression under UV-light. At 8 dpi, the plants inoculated with pTVBMV-GFP showed vein clearing symptoms similar to those of TVBMV-HN39 in the systemically infected leaves (Fig. 4B), indicating that GFP insertion did not affect virus infection and movement significantly. Green fluorescence was observed in and around the mid-vein and minor veins of the systemically infected leaves. By 14 dpi, these leaves showed strong green fluorescence in the systemically infected leaves, implying that GFP can be expressed stably to a high level (Fig. 4B). Virus species in the genus Potyvirus (family Potyviridae) are divided to several phylogenetic subgroups based on phylogenetic analyses (Adams et al., 2012). TVBMV is placed in the same group with Chronic bee paralysis virus, Wild tomato mosaic virus, Chilli veinal mottle virus and Pepper veinal mottle virus. To our knowledge, TVBMV is the first virus of this subgroup for which an infectious cDNA is now available. The P3-encoding sequence in several
R. Gao et al. / Virus Research 169 (2012) 276–281
279
Fig. 3. Symptoms and virus accumulation in N. benthamiana plants inoculated with pTVBMV and two mutants pTVBMVR182I and pTVBMVD198K . (A) Following inoculation with pTVBMV, the systemically infected leaf displays apparent vein clearing symptoms, whereas mild or no symptoms are observed in the leaves of plants inoculated with pTVBMVR182I and pTVBMVD198K 8 days post-inoculation. (B) Accumulation of TVBMV in the upper non-inoculated leaves of N. benthamiana plants at various times post inoculation, as determined by PTA-ELISA. (C) Detection of TVBMV RNA in the systemically infected leaves by qRT-PCR at 11 days post-inoculation. Levels of actin transcripts in these tissues were used as an internal control.
potyviruses is claimed to contain cryptic promoters that are active in bacterial cells and drive expression of proteins toxic to bacteria (Subr et al., 2000). This problem can be solved by insertion of intron(s) in the viral clones (Johansen, 1996; Johansen and Lund,
2008; López-Moya and García, 2000; Olsen and Johansen, 2001; Yamshchikov et al., 2001). For example, insertion of an intron within the P3-encoding region stabilized the infectious clone of Plum pox virus (López-Moya and García, 2000), and insertion of
Fig. 4. Expression of GFP using the vector pTVBMV-GFP GFP fluorescence was observed under UV light in the N. benthamiana leaves systemically infected with pTVBMV-GFP at 8 and 14 days post-inoculation (dpi).
280
R. Gao et al. / Virus Research 169 (2012) 276–281
introns into the P1, P3, CI and 6K2 encoding regions stabilized the full length clone of Pea seed-borne mosaic virus in E. coli (Olsen and Johansen, 2001). In our study, insertion of an intron into the P3-encoding sequence alone was not sufficient to stabilize the full length clone of TVBMV, but two additional introns in the CIencoding sequence stabilized the viral cDNA. Similar results have been obtained when introns were inserted in the P3- and CIencoding regions of the clone PVY-N605(123) (Bukovinszki et al., 2007). Site-directed mutagenesis of the infectious clone showed that the FRNK and CDN motifs of HC-Pro are involved in virulence of TVBMV. When the lysine was substituted for aspartic acid in the CDN motif of TVBMV HC-Pro, the virus became less virulent and the mutant virus accumulated to lower levels in infected plants than the wild type virus. The CDN motif has been reported to play a role in virus accumulation and symptom development also in other potyviruses (Desbiez et al., 2010; Gal-On, 2000; Hu et al., 2009; Torres-Barceló et al., 2008; Yambao et al., 2008). Mutation of the HC-Pro FRNK motif to FINK reduced both virulence and accumulation of TVBMV in plants, whereas a similar mutation in HC-Pro in Zucchini yellow mosaic virus has no such effect (Shiboleth et al., 2007). We also demonstrated with the construct pTVBMV-GFP that TVBMV has potential in use as a gene expression vector in plants. Stability of the recombinant viruses containing foreign genes often appears as a bottleneck for the applications (Chung et al., 2007), but our data indicate that pTVMBV-GFP is stable. Taken together, we have successfully constructed an infectious cDNA clone of TVBMV, which can be easily and cost-effectively delivered into plants with a portable biolistic device (HandyGun) that can be prepared from commonly available parts (Sikorskaite et al., 2010). The infectious TVBMV clone enables us to study virulence of TVBMV in plants, engineer mild strains for use in crossprotection against severe strains of TVBMV, and develop vectors for expression of foreign proteins in plants. Acknowledgments This study was supported by Special Research Funds for Doctoral Program (SRFDP, 20080434006; 20123702110013) from Ministry of Education, China, grants from National Natural Science Foundation of China (NSFC; 30971895 and 31011130031) and the Academy of Finland (1134759 and 1253126). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2012.07.010. References Adams, M.J., Zerbini, F.M., Stenger, D.C., Rabenstein, D., French, R., Valkonen, J.P.T., 2012. Family – Potyviridae. In: Andrew, M.Q.K., Elliot, L., Michael, J.A., Carstens, E.B. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses: The Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, pp. 1069–1089. Bukovinszki, Á., Götz, R., Johansen, E., Maiss, E., Balázs, E., 2007. The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains. Virus Research 127, 122–125. Caldo, R.A., Nettleton, D., Wise, R.P., 2004. Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16, 2514–2528. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802–805. Chen, X.-Z., Wen, L., Wei, D., Gao, R., Yuan, C.Y., Li, X.-D., 2009. Detection of viruses infecting tobacco in Mengyin. Shandong Agricultural Sciences 10, 62–63. Chung, B.N., Canto, T., Palukaitis, P., 2007. Stability of recombinant plant viruses containing genes of unrelated plant viruses. Journal of General Virology 88, 1347–1355.
Chung, B.Y.W., Miller, W.A., Atkins, J.F., Firth, A.E., 2008. An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences of the United States of America 105, 5897–5902. Desbiez, C., Girard, M., Lecoq, H., 2010. A novel natural mutation in HC-Pro responsible for mild symptomatology of Zucchini yellow mosaic virus (ZYMV, Potyvirus) in cucurbits. Archives of Virology 155, 397–401. Fribourg, C.E., Nakashima, J., 1984. Characterization of a new potyvirus from potato. Phytopathology 74, 1363–1369. Gal-On, A., 2000. A point mutation in the FRNK motif of the Potyvirus Helper Component-protease gene alters symptom expression in cucurbits and elicits protection against the severe homologous virus. Phytopathology 90, 467–473. González, J.M., Pénzes, Z., Almazán, F., Calvo, E., Enjuanes, L., 2002. Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. Journal of Virology 76, 4655–4661. Holzberg, S., Brosio, P., Gross, C., Pogue, G.P., 2002. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant Journal 30, 315–327. Hu, X., Meacham, T., Ewing, L., Gray, S.M., Karasev, A.V., 2009. A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco. Virus Research 143, 68–76. Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. The Journal of General Virology 78, 3141–3145. Johansen, I.E., 1996. Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo. Proceedings of the National Academy of Sciences of the United States of America 93, 12400–12405. Johansen, I.E., Lund, O., 2008. Insertion of introns: a strategy to facilitate assembly of infectious full length clones. In: Foster, G., Johansen, I.E., Hong, Y., Nagy, P. (Eds.), Plant Virology Protocols, vol. 451. Humana Press, pp. 535–544. Kelloniemi, J., Mäkinen, K., Valkonen, J.P.T., 2008. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Research 135, 282–291. Lacomme, C., Hrubikova, K., Hein, I., 2003. Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. The Plant Journal 34, 543–553. Lan, Y.-F., Liu, J.-L., Gao, R., Wang, H.-Y., Zhu, T.-S., Zhu, X.-P., Li, X.-D., 2007. Expression of Tobacco vein banding mosaic virus coat protein in E. coli and preparation of antiserum. Acta Phytopathologica Sinica 37, 461–466 (in Chinese). Lindbo, J.A., 2007. TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiology 145, 1232–1240. Liu, H., Naismith, J.H., 2008. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology 8, 91. López-Moya, J.J., García, J.A., 2000. Construction of a stable and highly infectious intron-containing cDNA clone of Plum pox potyvirus and its use to infect plants by particle bombardment. Virus Research 68, 99–107. Monger, W.A., Seal, S., Isaac, A.M., Foster, G.D., 2001. Molecular characterization of the Cassava brown streak virus coat protein. Plant Pathology 50, 527–534. Nagyová, A., Subr, Z., 2007. Infectious full-length clones of plant viruses and their use for construction of viral vectors. Acta Virologica 51, 223–237. Olsen, B.S., Johansen, I.E., 2001. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus. Archives of Virology 146, 15–25. Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C., 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant Journal 25, 237–245. Sainsbury, F., Canizares, M.C., Lomonossoff, G.P., 2010. Cowpea mosaic virus: the plant virus-based biotechnology workhorse. Annual Review of Phytopathology 48, 437–455. Scotto-Lavino, E., Du, G., Frohman, M.A., 2007. 5 end cDNA amplification using classic RACE. Nature Protocols 1, 2555–2562. Shiboleth, Y.M., Haronsky, E., Leibman, D., Arazi, T., Wassenegger, M., Whitham, S.A., Gaba, V., Gal-On, A., 2007. The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. Journal of Virology 81, 13135–13148. Sikorskaite, S., Vuorinen, A.L., Rajamäki, M.-L., Nieminen, A., Gaba, V., Valkonen, J.P.T., 2010. HandyGun: an improved custom-designed, non-vacuum gene gun suitable for virus inoculation. Journal of Virological Methods 165, 320–324. Simón-Buela, L., Guo, H.S., García, J.A., 1997. Long sequences in the 5 noncoding region of Plum pox virus are not necessary for viral infectivity but contribute to viral competitiveness and pathogenesis. Virology 233, 157–162. Subr, Z., Fomitcheva, V.W., Ühne, T.K., 2000. The complete nucleotide sequence of RNA1 of Barley mild mosaic virus (ASL1) and attempts at bacterial expression of the P3 protein. Journal of Phytopathology 148, 461–467. Tian, Y.P., Liu, J.L., Yu, X.Q., Lei, L.P., Zhu, X.P., Valkonen, J.P.T., Li, X.D., 2007. Molecular diversity of Tobacco vein banding mosaic virus. Archives of Virology 152, 1911–1915. Torres-Barceló, C., Martín, S., Daròs, J.-A., Elena, S.F., 2008. From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro. Genetics 180, 1039–1049. Ülper, L., Sarand, I., Rausalu, K., Merits, A., 2008. Construction, properties, and potential application of infectious plasmids containing Semliki Forest virus full-length cDNA with an inserted intron. Journal of Virological Methods 148, 265–270. Wang, H.Y., Zhu, T.S., Cui, T.T., Hou, S.S., Yin, X., Li, X.-D., Lei, L.P., Zhu, X.P., 2010. Complete genome sequence of a tobacco isolate of the Tobacco vein banding mosaic virus strain prevailing in China. Archives of Virology 155, 293–295. Yambao, M.L.M., Yagihashi, H., Sekiguchi, H., Sekiguchi, T., Sasaki, T., Sato, M., Atsumi, G., Tacahashi, Y., Nakahara, K.S., Uyeda, I., 2008. Point mutations in helper
R. Gao et al. / Virus Research 169 (2012) 276–281 component protease of Clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean. Archives of Virology 153, 105–115. Yamshchikov, V., Mishin, V., Cominelli, F., 2001. A new strategy in design of (+)RNA virus infectious clones enabling their stable propagation in E. coli. Virology 281, 272–280. Yang, S.J., Revers, F., Souche, S., Lot, H., Le Gall, O., Candresse, T., Dunez, J., 1998. Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects
281
of intron-insertion on their viability in Escherichia coli and on their infectivity to plants. Archives of Virology 143, 2443–2451. Yu, X.-Q., Lan, Y.-F., Wang, H.-Y., Liu, J.-L., Zhu, X.-P., Valkonen, J.P.T., Li, X.-D., 2007. The complete genomic sequence of Tobacco vein banding mosaic virus and its similarities with other potyviruses. Virus Genes 35, 801–806. Zhang, C.-L., Gao, R., Wang, J., Zhang, G.-M., Li, X.-D., Liu, H.-T., 2011. Molecular variability of Tobacco vein banding mosaic virus populations. Virus Research 158, 188–198.