Journal of General Virology (1997), 78, 11811185. Printed in Great Britain ...............................................................................................................................................................................................................

SHORT COMMUNICATION

Plasmid vector for cloning infectious cDNAs from plant RNA viruses : high infectivity of cDNA clones of tomato aspermy cucumovirus
Bu-Jun Shi, Shou-Wei Ding and Robert H. Symons
Department of Plant Science, Waite Institute, University of Adelaide, Glen Osmond, SA 5064, Australia

An improved version of the previously obtained cloning vector pCass was constructed by partially duplicating the 35S promoter used to drive the transient transcription of cloned viral cDNAs. Fulllength cDNAs of the three genomic RNAs of tomato aspermy cucumovirus (TAV) cloned in this improved pCass (designated pCass2) gave a 3-fold higher infectivity in two plant species tested than the same cDNAs cloned in pCass1 with only a single 35S promoter. Host range, symptoms, morphology of viral particles and viral progeny RNAs induced by these sets of infectious cDNA clones analysed were identical to those induced by the wild-type virus. A mutant of genomic TAV RNA 3 containing a 163 nt deletion in the 3h untranslated region was stably maintained in the progeny RNAs, indicating that these cDNA clones may facilitate a study of virus function. This is the rst report of infectious cDNA clones of TAV as well as of infectious cDNA clones with a duplicated 35S promoter of CaMV.

The genus Cucumovirus contains three virus species, cucumber mosaic virus (CMV), tomato aspermy virus (TAV) and peanut stunt virus (PSV), all economically important plant pathogens (Palukaitis et al., 1992). Member viruses of this genus have a tripartite single-stranded RNA genome of messenger sense, similar to the other three genera (Bromovirus, Alfamovirus and Ilarvirus) of the Bromoviridae (Murphy et al., 1995). We have recently reported that the cucumoviruses encode a small overlapping gene 2b in addition to the four major genes (1a, 2a, 3a and coat protein) common to the four genera (Ding et al., 1994 ; Shi et al., 1997 a). Analyses of the recently published sequence data suggested that the ilarviruses
Author for correspondence : Bob Symons. Fax j61 8 8303 7102. e-mail bsymons!waite.adelaide.edu.au Present address : Institute of Molecular Agrobiology, National University of Singapore, Singapore, 59A, The Fleming, Science Park Drive, Singapore 118240.

also encode a cucumoviral 2b-like gene (Ding et al., 1995 b ; unpublished results). Mutational analyses have shown that the 2b gene of the Q strain of CMV encodes a host-specic long-distance virus movement function (Ding et al., 1995 b). Recombinant assays showed that the 2b gene of V-TAV can functionally substitute for that of Q-CMV in at least six host species shared by the parental viruses (Ding et al., 1996) but not in cucumber (unpublished results), which is a systemic host for Q-CMV but immune to V-TAV (Habili & Francki, 1974). To further study the host specicity controlled by dierent cucumovirus genes, it is essential to establish an ecient infection system for the cloned viral genomes of several representative species. Such a system is presently available only for strains of CMV, the type species of the genus (Hayes & Buck, 1990 ; Rizzo & Palukaitis, 1990 ; Suzuki et al., 1991 ; Boccard & Baulcombe, 1992 ; Zhang et al., 1994 ; Ding et al., 1995 a). We describe here the infectious full-length cDNA clones of TAV constructed in a modied pCass vector (pCass2) as well as in a pUC19-based pCass (pCass1). The original pCass vector was based on the plasmid pSP72 (Ding et al., 1995 a). We show that the infectivity of TAV cDNAs controlled under a partially duplicated CaMV 35S promoter in pCass2 is 3-fold higher than the same cDNAs driven by the single 35S promoter in pCass1. Purication of V-TAV virions and viral RNAs was as described for CMV (Peden & Symons, 1973). The nucleotide (nt) sequences of the three genomic RNAs of V-TAV have been determined (Moriones et al., 1991 ; Bernal et al., 1991 ; F. Garc! a-Arenal, personal communication). First-strand cDNAs to the three V-TAV genomic RNAs were prepared as described previously for Q-CMV (Ding et al., 1995 a). Second-strand synthesis followed the protocol in Sambrook et al. (1989) using RNaseH, Escherichia coli DNA polymerase I and E. coli DNA ligase, and cloning the resultant double-stranded DNAs into the SmaI site of pBluescript SK(j). The near full-length cDNA clones selected for each of three RNAs, together with the missing 5h-terminal sequences obtained by PCR, were used for assembling the three full-length cDNA clones in pCass1 or pCass2 using a general strategy devised previously (Ding et al., 1995 a). pCass1 was obtained by excising the 35S promoter

0001-4568 # 1997 SGM

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B.-J. Shi, S.-W. Ding and R. H. Symons

(a) (b)

(c)

Fig. 1. Structures of the cloning vector pCass2 and infectious full-length cDNA clones of V-TAV genomic RNAs, 1, 2 and 3. (a) Molecular structure of pCass2. The expression cassette including the 35S promoter (P) and terminator (T) was rst transferred from pCass to pUC19 in which the polycloning restriction sites from EcoRI to SmaI had been eliminated in advance, and the partial duplication of the 35S P achieved using the unique EcoRV site at position k90 as described (Kay et al., 1987). The insertion site between StuI and SacI for the full-length cDNAs of V-TAV RNAs 1, 2 and 3 is indicated. The StuI site from which transcription starts is methylation sensitive and therefore pCass2 should be propagated in dcm strains of E. coli, such as JM110. (b) Schematic representation of the three genomic cDNA clones of V-TAV. 35S P, 35S T and each of the genomic RNAs with sizes are shown. The position for excision of the expression cassette from each of the plasmid cDNA clones is indicated by arrows with the relevant restriction enzyme. Tandem repeats at the 3h end of genomic cDNA 3 are indicated by a duplicated arrow. (c) Schematic representation of genomic cDNA 3 mutant in which one of the tandem repeats was deleted.

and the 35S terminator from pCass and assembling them in the modied pUC19. The 35S promoter in pCass1 was partially duplicated from nt k410 to k90 as described (Kay et al., 1987) to yield pCass2 (Fig. 1 a). This partial duplication resulted in at least a 10-fold increase in expression levels of foreign genes in transgenic plants (Kay et al., 1987). The three cDNA clones corresponding to TAV RNAs 1, 2 and 3 constructed in pCass1 were designated pCass1T1, pCass1T2 and pCass1T3 respectively and those in pCass2 were designated pCass2T1, pCass2T2 and pCass2T3, respectively (Fig. 1 b). The full-length cDNA clone of each of three genomic RNAs was puried by Superose 6 gel ltration chromatography (Skingle et al., 1990) and partially sequenced at the 5hand 3h-terminal regions to conrm the integrity of the promoter fusions and identity to the published sequences. Several variations were observed in the 3h untranslated regions of the cDNAs 1 and 3 as compared to the published sequences of the same strain of TAV (Bernal et al., 1991 ; F. Garc! a-Arenal, personal communication). The variations included one insertion (ATT between nt 3312 and 3313) in cDNA 1 and two substitutions (G"*(% to T and T"*(& to G) in cDNA 3 and one T insertion between nt 2369 and 2371 of cDNA 3. Importantly, the insert (ATT) in cDNA 1 creates a recognition site for EcoRI which was conrmed by digestion with this enzyme. For assaying infectivity, each of the puried three genomic plasmid cDNAs was digested with a restriction endonuclease (HaeII for RNAs 1 and 3 clones and PvuII for RNA 2 clones) to

release the cloned insert, a step previously shown to enhance infectivity of cDNA clones of CMV containing a single 35S promoter (Ding et al., 1995 a). Equal amounts (10 g per plant of each of the digested three genomic plasmid cDNAs in 10 l of water) were inoculated on Nicotiana glutinosa plants which had been in the dark for 24 h. Both sets of the V-TAV cDNA clones were infectious and produced systemic virus infections indistinguishable from each other or from infections using virions as judged by symptomology (Fig. 2 a), particle morphology (not shown), ethidium bromide staining (EB, Fig. 2 b) and Northern blot analysis (Fig. 2 c) of virion RNAs. These results further show the usefulness of pCass as a general cloning vector for constructing infectious cDNA clones of plant RNA viruses. RNA 4A (Fig. 2 b, c), rst designated in CMV by Peden & Symons (1973), is a subgenomic RNA generated from the 3hterminal 702 nt of TAV RNA 2 for expressing a 2b protein encoded by RNA 2 (Shi et al., 1997 a). TAV RNAs 3B and 5 (Fig. 2 b, c) are a novel class of subgenomic RNAs which encode no open reading frame of 21 codons or longer. Both RNAs 3B and 5 are derived from the 3h-terminal 486 and 323 nt of RNA 3, respectively (Shi et al., 1997 b). In contrast to RNAs 3B and 5, only very low levels of RNA 4A were encapsidated in the viral particles (Fig. 2 b, c), indicating varying specicity of in vivo encapsidation of V-TAV RNAs. The 5h-terminal 80 nt or so of the three progeny viral RNAs isolated both from N. glutinosa and N. clevelandii plants

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Infectious full-length cDNA clones of TAV

(a)

(b)

(c)

Fig. 2. Viral RNAs in N. glutinosa plants inoculated with either cDNA clones or virions of V-TAV. (a) Systemic mosaic symptoms were induced with pCass2T1T2T3 (left top) or pCass1T1T2T3 (right top) or the wild-type virions (left bottom) or pCass2T1T2T3(163) (right bottom) by manual inoculation. A control healthy N. glutinosa plant with mock-inoculation is shown in the middle on the right. (b) Electrophoresis analysis on a 1n2 % agarose gel stained with ethidium bromide (EB) of encapsidated RNAs extracted from N. glutinosa inoculated with the wild-type V-TAV (lane 1), pCass2T1T2T3 (lane 2) or pCass1T1T2T3 (lane 3). (c) Northern blot analysis of encapsidated RNAs. Virion RNAs extracted from N. glutinosa inoculated with the wild-type V-TAV (lane 4), pCass2T1T2T3 (lane 5) or pCass1T1T2T3 (lane 6) were electrophoresed on a 1n2 % agarose gel containing 1n1% formaldehyde, transferred onto a Hybond-Nj membrane (Amersham) and hybridized with a strandspecic RNA probe complementary to the 3h-terminal 128 nt of all three genomic RNAs. The positions of viral RNAs 1, 2, 3, 4, 4A, 3B and 5 are indicated, of which RNA 3B is a newly discovered subgenomic RNA derived from RNA 3 (Shi et al., 1997 b).

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B.-J. Shi, S.-W. Ding and R. H. Symons

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(b)

Fig. 3. Dose response of plants to pCass1T1T2T3 and pCass2T1T2T3 45 days after inoculation. (a) N. glutinosa seedlings (two true leaves) were inoculated with pCass1T1T2T3 and pCass2T1T2T3. All points are the average of the results of two separate experiments with 15 plants each. (b) N. clevelandii seedlings (two true leaves) were inoculated with pCass1T1T2T3 and pCass2T1T2T3.

infected with pCass1T1T2T3 (the mixture of three genomic cDNA clones under the control of the single promoter) or pCass2T1T2T3 (the mixture of three genomic cDNA clones under the control of the partially duplicated promoter) as determined by RNA dideoxynucleotide sequencing (Ding et al., 1995 b) were all identical to each other as well as to those of the respective plasmid DNA inocula with one following exception. One U insertion was found between nt 2 and 3 of both progeny RNAs 1 and 2 derived from pCass2T1T2T3. This extra U has been persistently maintained in several subsequent passages and had no apparent eect on symptomology. It is not clear how this insertion occurred or whether it is related to the partial duplication of the CaMV 35S promoter. The relative infectivity of the V-TAV cDNA clones constructed in pCass1 and pCass2 was examined in two Nicotiana species, N. glutinosa and N. clevelandii. Each of the 13 dilutions from either set of the TAV cDNA clones was inoculated onto 30 seedlings (two replicates of 15 plants each) of either plant species and the percentages of plants infected were recorded 45 days after inoculation. The following points are evident from the data presented in Fig. 3. (i) When using lower amounts of plasmids as inocula the relative infectivity of

the pCass2-based TAV cDNA clones was clearly higher (3fold) than that of the pCass-based clones ; however, the dierence was not obvious for higher amounts of plasmid DNAs as inocula. It is most likely that the relatively higher infectivity of pCass2-based TAV clones resulted from a higher level of transient transcription as directed by the partially duplicated promoter inside the inoculated cells. It should be pointed out that our data do not exclude the extra U found in RNAs 1 and 2 derived from pCass2-based clones being the cause of higher infectivity. (ii) There is an obvious dierence in susceptibility to virus infection from plasmid inocula between the two host species. For the same amounts, the percentage of N. clevelandii plants which became infected is always higher than that of N. glutinosa plants. (iii) Low amounts of plasmid DNAs (0n2 to 2 g each of the three plasmids per plant) were sucient to achieve an infection rate over 50 %. Thus, a largescale plasmid DNA preparation can generate a substantial amount of virus inoculum. Further infectivity assays using pCass2T1T2T3 demonstrated that the plasmid DNAs as inocula were as eective as viral particles on six additional host species : Chenopodium amaranticolor, Datura stramonium, Gomphrena globosa, Lycopersicon esculentum, Physalis oridana and N. tabacum cv. White Burley. However, up to 45 g of pCass2T1T2T3 (45 g each of pCass2T1, pCass2T2 and pCass2T3) failed to infect cucumber, which is a non-host of V-TAV (Habili & Francki, 1974). Thus, our data indicate that the host range of the plasmid inocula reected the true host range of V-TAV when nine plant species were examined. We have recently characterized a novel subgenomic RNA 3B associated with V-TAV (Shi et al., 1997 b). This RNA is 486 nt long and identical in sequence to the 3h 486 nt of RNA 3. RNA 3B consists of three characteristic regions, two tandem repeats of 163 nt each at the 5h terminus and the 3h-terminal sequence of 160 nt which is highly conserved among all TAV RNAs characterized to date. A mutant of the RNA 3 cDNA clone (pCass2T3) was constructed by deleting one such repeat and was combined with the wild-type RNAs 1 and 2 cDNA clones to inoculate seedlings of N. glutinosa and N. clevelandii in addition to the above six host species. Northern blot analysis of RNAs from the inoculated plants showed the accumulation of all known V-TAV RNAs and the expected decrease in size of RNAs 3, 4 and 3B, thus conrming that RNA 3B was derived from RNA 3 (Shi et al., 1997 b). However, no dierence was observed between the wild-type and mutant viruses in symptomology (data not shown) and host range, suggesting a non-essential role for the additional copy of the tandem repeat that is absent in strains B, C and P of TAV (OReilly et al., 1991, 1994 ; Salanki et al., 1994). Ecient infection was achieved previously using the fulllength cDNAs of CMV genomic RNAs cloned in a new plasmid vector, pCass (Ding et al., 1995 a). We describe here a signicantly improved version of pCass by partially duplicating the 35S promoter controlling the transcription of cloned

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Infectious full-length cDNA clones of TAV

viral cDNAs. An increase in the relative infectivity was evident for the full-length TAV cDNAs cloned in pCass2 over the same cDNAs cloned in pCass1. These infectious cDNA clones of CMV (Ding et al., 1995 a) or of TAV (in this study) have been used successfully for mutational analysis of the virus genome, constructing inter- or intra-species hybrid viruses, reconstituting pseudorecombinants between the two viruses and as the helper for satellite RNAs (Ding et al., 1995 a, b, 1996 ; Shi et al., 1997 b). Compared with other strategies currently used to obtain progeny virus from cDNA clones such as infectious RNAs transcribed in vitro from cDNA clones (Boyer & Haenni, 1994) and particle bombardment of cDNA clones (Gal-On et al., 1995 ; Fakhfakh et al., 1996), the pCass2 system we developed oers several unique features including easy preparation of plasmid inocula and plant infection by conventional mechanical inoculation, and thus is simple to use and cost-eective.
The authors wish to thank Fernando Garc! a-Arenal for kindly supplying us with the unpublished sequence of V-TAV and Wanxiang Li for technical help. This work was supported by the Australian Research Council Special Research Centre for Basic and Applied Plant Molecular Biology. B.-J. S. is the recipient of a University of Adelaide Overseas Postgraduate Research Scholarship.

Habili, N. & Francki, R. I. B. (1974). Comparative studies on tomato

aspermy and cucumber mosaic viruses. III. Further studies on relationship and construction of a virus from parts of the two viral genomes. Virology 61, 443449. Hayes, R. J. & Buck, K. W. (1990). Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from complementary DNA amplied using the polymerase chain reaction. Journal of General Virology 71, 25032508. Kay, R., Chan, A., Daly, M. & McPherson, J. (1987). Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236, 12991302. Moriones, E., Roossinck, M. J. & Garc! a-Arenal, F. (1991). Nucleotide sequence of tomato aspermy virus RNA 2. Journal of General Virology 72, 779783.
Murphy, F. A. , Fauquet, C. M., Bishop, D. H. L., Ghabrial, S. A., Jarvis, A. W., Martelli, G. P., Mayo, M. A. & Summers, M. D. (editors) (1995).

Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Vienna & New York : Springer-Verlag. OReilly, D., Thomas, C. J. R. & Coutts, R. H. A. (1991). Tomato aspermy virus has an evolutionary relationship with other tripartite RNA plant viruses. Journal of General Virology 72, 17. OReilly, D., Thomas, C. J. R. & Coutts, R. H. A. (1994). Nucleotide sequence of RNA 3 of the British type isolate (Blencowe strain) of tomato aspermy virus. Virus Genes 8, 7981.
Palukaitis, P., Roossinck, M. J., Dietzgen, R. G. & Francki, R. I. B. (1992). Cucumber mosaic virus. Advances in Virus Research 41, 281348. Peden, K. W. C. & Symons, R. H. (1973). Cucumber mosaic virus

References
Bernal, J. J., Moriones, E. & Garc! a-Arenal, F. (1991). Evolutionary relationships in the cucumoviruses : nucleotide sequence of tomato aspermy virus RNA 1. Journal of General Virology 72, 21912195. Boccard, F. & Baulcombe, D. C. (1992). Infectious in vitro transcripts from amplied cDNAs of the Y and Kin strains of cucumber mosaic virus. Gene 114, 223227. Boyer, J. C. & Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses. Virology 198, 415426. Ding, S.-W., Anderson, B. J., Haase, H. R. & Symons R. H. (1994). New overlapping gene encoded by the cucumber mosaic virus genome. Virology 198, 593601. Ding, S.-W., Rathjen, J. P., Li, W.-X., Swanson, R., Healy, H. & Symons, R. H. (1995 a). Ecient infection from cDNA clones of cucumber mosaic

cucumovirus RNAs in a new plasmid vector. Journal of General Virology 76, 459464. Ding, S.-W., Li, W.-X. & Symons, R. H. (1995 b). A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. EMBO Journal 14, 57625772. Ding, S.-W., Shi, B.-J., Li, W.-X. & Symons, R. H. (1996). An interspecies hybrid RNA virus is signicantly more virulent than either parental virus. Proceedings of the National Academy of Sciences, USA 93, 74707474. Fakhfakh, H., Vilaine, F., Makni, M. & Robaglia, C. (1996). Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y. Journal of General Virology 77, 519523.
Gal-On, A., Meiri, E., Huet, H., Hua, W. J., Raccah, B. & Gaba, V. (1995). Particle bombardment drastically increases the infectivity of

contains a functionally divided genome. Virology 53, 487492. Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length complementary DNA clones of cucumber mosaic virus RNA 1, 2 and 3 : Generation of infectious RNA transcripts. Molecular and General Genetics 222, 249256. Salanki, K., Balazs, E. & Burgyan, J. (1994). Nucleotide sequence and infectious in vivo transcripts of RNA 3 of tomato aspermy virus pepper isolate. Virus Research 33, 281289. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory. Shi, B.-J., Ding, S.-W., & Symons, R. H. (1997 a). In vivo expression of an overlapping gene encoded by the cucumoviruses. Journal of General Virology 78, 237241. Shi, B.-J., Ding, S.-W., & Symons, R. H. (1997 b). Two novel subgenomic RNAs derived from RNA 3 of tomato aspermy cucumovirus. Journal of General Virology 78, 505510. Skingle, D. C., Mclnnes, J. L. & Symons, R. H. (1990). An improved method for eliminating RNA contamination of plasmid DNA preparations. BioTechniques 9, 314317.
Suzuki, M., Kuwata, S., Masuta, C., Nitta, N. & Takanami, Y. (1991).

Functional analysis of deletion mutants of cucumber mosaic virus RNA 3 using an in vitro transcription system. Virology 183, 106113. Zhang, L., Hanada, K. & Palukaitis, P. (1994). Mapping local and systemic symptom determinants of cucumber mosaic cucumovirus in tobacco. Journal of General Virology 75, 31853191.

cloned DNA of zucchini yellow mosaic potyvirus. Journal of General Virology 76, 32233227.

Received 27 November 1996 ; Accepted 14 January 1997

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