Movement of a Barley Stripe Mosaic Virus Chimera with a Tobacco Mosaic Virus Movement Protein

VIROLOGY

217, 435–441 (1996) 0137

ARTICLE NO.

Movement of a Barley Stripe Mosaic Virus Chimera with a Tobacco Mosaic Virus Movement Protein A. G. SOLOVYEV, D. A. ZELENINA, E. I. SAVENKOV, V. Z. GRDZELISHVILI, S. YU. MOROZOV, D.-E. LESEMANN,* E. MAISS,*,1 R. CASPER,* and J. G. ATABEKOV2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia; and *Institute of Biochemistry and Plant Virology, Biologische Bundesanstalt fu¨r Land- and Forstwirtschaft, Messeweg 11/12, D-38104 Braunschweig, Germany Received September 28, 1995; accepted January 12, 1996 The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNAb replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs a and g, together with the hybrid RNAb transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves of Nicotiana benthamiana and Chenopodium amaranticolor, which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions on C. amaranticolor were influenced by the foreign MP gene. However, the hybrid virus failed to infect barley, N. tabacum (var. Samsun), and N. clevelandii, the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-tocell movement in a host-dependent manner. q 1996 Academic Press, Inc.

INTRODUCTION

and Hamilton, 1991; Richins et al., 1993). In particular, there was complementation of tobacco mosaic virus (TMV) transport in barley by barley stripe mosaic virus (BSMV) (Hamilton and Dodds, 1970), and the transport of BSMV was facilitated by TMV in tobacco (Malyshenko et al., 1989). The rod-shaped BSMV has a tripartite genome consisting of three RNAs designated a, b, and g. RNAb contains four ORFs, the 5*-proximal coat protein (CP) gene, and the TGB, collectively encoding the TF. The CP is dispensable for the replication and systemic movement of BSMV, whereas mutations in the TGB eliminate movement (Petty and Jackson, 1990). Here, we replaced the TGB in BSMV ND18 RNAb with the TMV 30K MP gene and assayed the ability of the resulting hybrid to infect the host and nonhost plants of the parental viruses.

The cell-to-cell translocation of a plant virus genome is a function of both the viral and the host genomes, in which virus-coded movement protein(s) (MPs) and hostcoded components are involved (for reviews, see Atabekov and Taliansky, 1990; Maule, 1991; Citovsky and Zambryski, 1993; Deom et al., 1992; Lucas and Gilbertson, 1994). The strategies for coding and expression of the cellto-cell transport function (TF) vary substantially between different virus groups. For example, tobamoviruses contain a single 30K MP gene in a unipartite genome (Deom et al., 1987). Unlike the single-MP-coding viruses, the genomes of hordeiviruses (Petty et al., 1990; Jackson et al., 1991), furoviruses (Gilmer et al., 1992), and potexviruses (Beck et al., 1991) contain the so-called triple gene block (TGB) (Morozov et al., 1989) and require three MPs to express their TF. Much evidence points to the conclusion that viruses belonging to different taxonomic groups are able to complement each other’s TF, i.e., that the MPs coded by unrelated plant viruses are functionally interchangeable (Ziegler-Graff et al., 1991; Taliansky et al., 1993; Fuentes

MATERIALS AND METHODS Plant material Nicotiana tabacum, N. benthamiana, and Chenopodium amaranticolor were grown in a greenhouse under natural light conditions supplemented with sodium halide lamps (16-hr photoperiod). Hordeum vulgare ‘‘Black Hulles’’ plants were maintained in growth chambers (16-hr light/8-hr dark periods at 257).

1 Present address: Institut fu¨r Pflanzenkrankheiten und Pflanzenschutz, University of Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany. 2 To whom correspondence and reprint requests should be addressed. Fax: (095) 938-06-01. E-mail: [email protected].

cDNA clones Full-length cDNA clones of the ND18 strain of BSMV capable of producing infectious in vitro transcripts were 0042-6822/96 $18.00

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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kindly provided by A. O. Jackson, University of California, Berkeley (Petty et al., 1988, 1989). First strand cDNA was synthesized from the TMV-U1 RNA using AMV reverse transcriptase (Promega) and the 3*-primer d(5*-AACACTCTTTACAGGGGTAAA) complementary to the TMV-U1 30K gene nucleotides 82–102 (positions 4984–4504 in TMV genome) (Goelet et al., 1982). The reaction mix (20 ml) contained 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 10 mM magnesium chloride, 1 mM dNTPs, 20 units of RNasin (Promega), and cDNA product was heat denatured at 957 for 5 min. The dsDNA was generated and amplified in a reaction containing 50 pmol of 5*-primer d(5*-GGGccatggCTCTAGTTGTTAAAGGAAAAG), which represents TMV 30K gene nucleotides 1–25 (positions 4903–4927 in TMV genome) and contains a unique 5*-flanking NcoI site (in lowercase), 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 5 mM magnesium chloride, and 3 units of Taq polymerase in a final volume of 60 ml. All PCRs were 20 cycles of 1 min at 947, 1 min at 537, and 2 min at 727. The resulting amplified DNA fragment, containing an internal BglII site (position 4967 in TMV genome) and 5*-flanking NcoI site, was isolated with the QIAEX gel extraction kit (QIAGEN). The TMV-U1 MP gene cDNA clone pKS-30K was previously generated to include flanking EcoRI and BamHI sites at the 5*- and 3*-termini, respectively (Ivanov et al., 1994).

daily for up to 15 days. The necrotic lesion symptoms were scored visually and recorded by photography. Detection of BSMV infection Leaf disks excised from individual leaves, or disks combined from five to six leaves, were ground in 50 mM sodium phosphate, pH 7 (0.2 ml buffer per 100 mg of tissue). The unfractionated homogenate was mixed with an equal volume of a 21 SDS–polyacrylamide gel electrophoresis sample buffer (Laemmli, 1970) followed by boiling for 2 min and fractionation in a 15% gel by PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher and Schuell) and detected with BSMV antiserum (kindly provided by V. K. Novikov). Immunosorbent electron microscopy (ISEM) (Milne and Lesemann, 1984) was adapted for analysis of BSMV particles in homogenates of C. amaranticolor leaves. The grids were treated for 5 min with antiserum to BSMV diluted 1:1000, incubated with extracts of local lesion tissues for 2 days, washed, and negatively stained with 1% uranyl acetate. For immunodecoration experiments, anti-BSMV serum (collection of BBA, Braunschweig) diluted 1:50, was used. Analysis of RNA

Infectious wt and chimeric BSMV transcripts were synthesized from full-length clones linearized with MluI for RNAs a and g, and with SpeI for wt and chimeric RNAb. Transcription with T7 RNA polymerase (Promega) and inoculation of plants were performed as described previously (Petty et al., 1989; Petty and Jackson, 1990). Following inoculation, plants were maintained in growth chambers. Symptom formation was observed

Total cellular RNA was extracted by SDS–phenol–LiCl mixture as described (Verwoerd et al., 1989). To perform slot-blot hybridizations, total RNA preparations were immobilized on a nylon membrane using ‘‘Slot Blot,’’ Model PR648 (Hoefer Scientific Instruments). Northern blot analysis was conducted as described previously (Solovyev et al., 1994). The filters were probed with 32P-labeled cDNA prepared with a random deoxyhexamers cDNA labeling kit (Boehringer) on the isolated cloned DNA fragments specific for the TMV MP gene or BSMV 58K MP and CP genes as described in the manufacturer’s protocol. To determine the nucleotide sequence of the progeny of chimeric genomic RNAs, the DNA was amplified from total RNA preparation by RT–PCR (see above). The first strand synthesis primer d(5*-CCCggtaccATCAGATTTGAATGATCTGA) was complementary to positions 3059– 3084 in the 3*-UTR of BSMV RNAb (Gustafson and Armour, 1986) and contained a flanking KpnI site (in lowercase letters), whereas the second strand primer d(5*GAATggtaccTTCCAGATGCCGAGGAAG) represented the 3*-end of the BSMV CP gene (positions 654–681) and also contained a KpnI site. The resulting PCR-generated DNA fragment was digested with KpnI and cloned into the KpnI site of the pBluescript II (SK) cloning vector (Stratagene). Complete sequencing of the virus-specific DNA inserts was performed by using the dideoxynucleotide method and Erase-a-Base kit (Promega).

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Generation of BSMV RNAb hybrid cDNA clones pND-MPT (Fig. 1) was created by substituting the TMV 30K gene for the BSMV TGB in a two-step cloning procedure. First, the PCR fragment containing the 5*-terminal part of the TMV 30K gene was digested with NcoI and BglII (Fig. 1) and ligated into an NcoI–BglII-cleaved BSMV RNAb cDNA clone (Petty and Jackson, 1990) to generate pND-30K5 (not shown). The pKS-30K (see above) was digested with BglII and BamHI to produce a DNA fragment containing most of the TMV MP gene. This fragment was ligated into the BglII-cleaved pND-30K5. The resulting clones were tested for the proper insert orientation by restriction enzyme mapping. The deletion mutant clone pND-D58 was made by digestion of the wt cDNA clone of RNAb with NcoI and SalI (Petty and Jackson, 1990), filling in the cohesive ends and recircularizing the plasmid by blunt-end ligation. In vitro transcription and RNA inoculation

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FIG. 1. (A) Constructing the chimeric clone ND-MPT and deletion mutant ND-D58. Coding regions of BSMV and TMV genomes are shown by open boxes; shaded box corresponds to the TMV MP gene. The BSMV RNAb-coded products are indicated. Restriction sites used for constructing derivatives of BSMV-ND18 RNAb are shown by small arrows (dotted arrows indicate sites introduced by cloning procedures). Size of in vitro transcripts of pND-MPT and pND-D58 is indicated. (B) Nucleotide sequences around the borders between the inserted TMV 30K MP gene (uppercase letters) and BSMV RNAb (lowercase letters). The polylinker nucleotides and BglII site are denoted by / and !, respectively. The N- and C-terminal amino acids of the TMV MP gene are shown below the nucleotide sequence.

RESULTS

Although the natural systemic hosts of BSMV are monocotyledonous grasses, in contrast to TMV, which has

dicotyledonous hosts, both viruses are able to systemically infect N. benthamiana (Jackson et al., 1991). In accordance with previous studies (Petty and Jackson, 1990; Petty et al., 1990, 1994), infectious transcripts of the wt BSMV-ND18 cDNA clones produced typical necrotic lesions on C. amaranticolor and systemic symptoms on N. benthamiana and H. vulgare ‘‘Black Hulles.’’ The mixture of RNAa and RNAg transcripts alone, or RNAa, RNAg, and the deletion mutant pND-D58 RNAb transcripts, infected neither C. amaranticolor nor N. benthamiana. The mutant pND-D58 with the N-terminal one-third of the 58K MP (bb protein) deleted was analogous to the b-2.0 mutant of Petty and Jackson (1990). On the other hand, when N. benthamiana leaves were inoculated with the chimeric RNAb (ND-MPT) transcripts in the presence of wt BSMV-ND18 RNAa and RNAg transcripts, the hybrid virus accumulated in the inoculated leaves to levels comparable to those of wt BSMV-ND18, as determined by Western blot analysis (Fig. 2). The symptoms of infection (mild chlorosis) induced on the inoculated leaves by the hybrid virus appeared later than in wt BSMV infection

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Replacement of the BSMV TGB with the TMV MP gene For construction of the recombinant clone pND-MPT, the cDNA fragment encompassing the TMV-U1 MP gene (starting with the initiation codon and ending with the terminator followed by a polylinker-derived 7-nt sequence) was inserted in two steps into an NcoI–BglIIcleaved BSMV-ND18 RNAb cDNA clone (Fig. 1). In the final construct (pND-MPT), the BSMV bb and bd genes were replaced by the TMV 30K gene that was followed, in a different reading frame, by the 3*-terminal part of the bc gene and the 3*-UTR of BSMV genome (Figs. 1A and 1B). The TMV-specific insertion in pND-MPT was confirmed for integrity by dideoxynucleotide sequencing. Cell-to-cell movement characteristics of the BSMV– TMV hybrid in N. benthamiana

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FIG. 2. Immunodetection of the BSMV coat protein in N. benthamiana leaves infected with one of the following: wt BSMV transcripts (wtBSMV); a mixture of wt BSMV RNA a and g transcripts with the hybrid RNAb transcript (ND-MPT); a mixture of wt BSMV RNA a and g transcripts with the transcripts of the RNAb deletion variant (NDD58). Mock indicates mock-inoculated leaves. Position of the coat protein (CP) from purified BSMV particles is indicated.

and were not observed on uninoculated leaves. In line with this, no BSMV CP was detected in the noninoculated leaves by Western blotting (data not shown). To examine hybrid virus RNA accumulation, total RNA preparations isolated from the N. benthamiana leaves inoculated with wt BSMV and ND-MPT were subjected to slot-blot and Northern blot hybridization analyses with TMV MP, BSMV bb, and CP gene-specific probes (Figs. 3 and 4). Supporting the results of Western blot analysis, the presence of the TMV 30K gene sequences was detected only in leaves inoculated with ND-MPT (Fig. 3) but not in the noninoculated leaves, whereas parental viruses BSMV-ND18 and TMV caused systemic infections. To rule out the cross-hybridization between wt and hybrid genomes, parallel hybridization reactions were performed with probes specific for the BSMV 58K protein and TMV 30K MP genes (Fig. 3). The results confirmed that the virus accumulation observed in ND-MPT-infected leaves was not due to contamination with wt BSMV RNAb (Fig. 3). It should be emphasized that in hybrid virus infections, only chimeric RNAb (which was shorter

FIG. 4. Northern blot hybridization with a TMV 30K gene-specific probe (A) and a BSMV CP gene-specific probe (B) of total RNA isolated from inoculated N. benthamiana (N.b.) and barley (H.v.) leaves. Abbreviations are as in Fig. 2. Purified brome mosaic virus (BMV) RNA3 (2117 nt) was taken as a size marker (lane M) and visualized by ethidium bromide staining. Arrow indicates the position of BMV RNA3.

than wt BSMV RNAb) was detected by Northern blotting with BSMV CP gene-specific probe (Fig. 4). Maintenance of the TMV MP gene insert in the chimeric RNA progeny It has been shown that the MP genes undergo sequence modifications in monocot- and dicot-specific bromovirus hybrids (Mise et al., 1993; Schneider and Allison, 1993). Total RNA extracted from the ND-MPT-inoculated leaves of N. benthamiana exhibiting a strong Western blot signal (Fig. 2) was reverse transcribed and PCR amplified. The entire 30K MP gene sequence within the chimeric RNAb was amplified with BSMV-specific primers. The DNA fragment encompassing the TMV MP gene flanking by the BSMV RNAb sequences (Fig. 1) was cloned and sequenced. Comparison of the sequences of ND-MPT progeny revealed two base substitutions leading to amino acid changes in the MP [Thr(18) to Ser, and Asn(228) to Ser] relative to the wild-type TMV-U1 sequence (Goelet et al., 1982). These mutations are unlikely to have a dramatic effect on the TMV MP function, because they occurred in nonconserved regions (Melcher, 1990). Inability of the hybrid virus to infect the parental virus differential hosts

FIG. 3. Slot-blot hybridization of BSMV-specific (probe for the 58K gene) and TMV-specific (probe for the 30K gene) sequences in inoculated leaves of N. benthamiana (N.b.) and barley (H.v.); trb and trMPT indicate in vitro transcripts of wt BSMV RNAb and ND-MPT RNAb, respectively, used as controls. Other abbreviations are as in Fig. 2.

Barley and the members of Solanaceae (N. tabacum var. Samsun, N. clevelandii) are differential hosts for infection by BSMV and TMV, respectively (Jackson et al., 1991; Dawson and Hilf, 1992). To examine the contribution of the TMV 30K MP gene to the host specificity, 96 barley, 40 N. tabacum, and 12 N. clevelandii plants were inoculated with ND-MPT transcripts. No BSMV CP was detected in barley, tobacco, and N. clevelandii leaves inoculated with ND-MPT, whereas wt BSMV-ND18 transcripts infected 60–70% of inoculated barley leaves, and ND-MPT transcripts infected 50% of inoculated N. benthamiana leaves.

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FIG. 5. Phenotype of local lesions elicited on C. amaranticolor by BSMV-ND18, TMV, and the hybrid virus ND-MPT. (A) wt BSMV-ND18 (4 dpi); (B) wt TMV-U1 (3 dpi); (C) ND-MPT (12 dpi).

Induction of the necrotic lesions on C. amaranticolor by the hybrid virus

infections. However, the proportion of shorter particles (modal length, 75–95 nm) was somewhat larger from infections by the hybrid virus than by wt BSMV (data not presented). This is consistent with the shorter size of the ND-MPT RNAb species in comparison to RNAb of the wt BSMV (Fig. 1).

BSMV-ND18 caused large necrotic lesions on C. amaranticolor by 4 days postinfection (dpi) (Fig. 5A; Petty et al., 1989, 1994) and complete collapse by 7–9 dpi. The ND-MPT-induced local lesions appeared by 10 dpi; the size and morphology of lesions in ND-MPT infection (Fig. 5C) differed from those produced by wt BSMV-ND18, being more similar to the wt TMV-induced lesions (Fig. 5B). The TMV local lesions were first visible by 2–3 dpi and continued to enlarge for at least 4 more days, whereas the ND-MPT-induced lesions did not enlarge. Unexpectedly, our attempts to passage the ND-MPT hybrid virus from the C. amaranticolor local lesions to healthy C. amaranticolor plants were unsuccessful, whereas the wt BSMV-ND18 could be readily passaged, producing necrotic reaction on C. amaranticolor. Moreover, no CP could be detected by Western blot analysis in the ND-MPT-specific lesions, in contrast to wt BSMVand TMV-specific necrotic spots (data not shown). The level of the signal in Northern slot-blot analysis with 30Kspecific probe was at least several orders of magnitude weaker in lesions containing the hybrid virus than that for wt TMV infection (data not shown), despite the similar sizes of TMV and ND-MPT lesions. Consistent with this, ISEM revealed that accumulation of the BSMV rod-like particles in the ND-MPT-induced lesions was approximately 1000–10,000 times lower than in the lesions induced by wt BSMV. It is important to note that particles were rarely detected in the C. amaranticolor leaves inoculated with the movement-deficient BSMV mutant NDD58 control. The virus particles in C. amaranticolor produced by chimeric constructs were strongly decorated by BSMV antiserum, and the particle length distribution was generally similar in the hybrid virus and wt BSMV

There is now ample evidence for the complementation of cell-to-cell TF of transport-deficient plant viruses by unrelated helper viruses in doubly infected plants (for review, see Atabekov and Taliansky, 1990). Taking into account that the genetic elements, other than MP genes, can affect the cell-to-cell movement and long-distance spread (Allison et al., 1988; Watanabe et al., 1987; Nejidat et al., 1991; Jackson et al., 1991; Gal-on et al., 1994; Deom et al., 1994; De Jong and Ahlquist, 1995) such traditional double-infection experiments cannot be interpreted unequivocally in terms of functional interchangeability of the MPs coded by the partner viruses. In this study, the recombinant BSMV was used to examine the functional interchangeability of cell-to-cell TF coded by two viruses having no sequence similarity between their MPs (BSMV and TMV). It can hardly be assumed that the biochemical functions of the TGB-coded proteins completely coincide with those coded by monocistronic transport genes with which TGB MPs do not have structural or sequence similarity. However, the BSMV TGB MPs can be replaced by a single TMV MP coded by the hybrid virus in the inoculated N. benthamiana leaves (Figs. 2–4). Unlike the parental viruses, the hybrid BSMV failed to infect N. benthamiana systemically. The inability of ND-MPT to systemically infect N. benthamiana might reflect some fundamental differences in the BSMV and TMV transport systems. For example,

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DISCUSSION

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it may concern the contribution of the TMV CP, but not BSMV CP, in long-distance movement (Dawson and Hilf, 1992; Jackson et al., 1991). Obviously, the failure of the hybrid virus to infect N. benthamiana systemically was not due to reduction of the rate of its accumulation in inoculated leaves, since the level of ND-MPT accumulation was similar to that of wt BSMV (Fig. 2). Although the ND-MPT recombinant directed sufficient cell-to-cell spread in inoculated leaves of N. benthamiana, it was unable to infect either barley or N. tabacum (var. Samsun) (and N. clevelandii), selective hosts of BSMV and TMV, respectively. These data indicate that in common hosts the MP may function independently of any other virus-encoded protein(s) to promote the hybrid virus cell-to-cell movement. However, in selective hosts some kind of host-specific adaptation of MP genes and, probably, other genomic elements (e.g., replicase gene) is needed for the hybrid virus cell-to-cell movement. In other words, the hybrid BSMV is presumably able to replicate and produces the TMV MP in primarily inoculated cells, yet nonetheless fails to move from cell to cell in the selective hosts. This seems to contradict the results of the movement complementation experiments in plants doubly inoculated with TMV and BSMV: the cell-to-cell movement of BSMV in barley was facilitated by TMV (Malyshenko et al., 1989), and TMV movement in barley was mediated by BSMV (Hamilton and Dodds, 1970). One might assume that in certain virus–host combinations the presence of an active MP in the cell per se is a necessary but insufficient condition to ensure an efficient cell-to-cell spread of the hybrid virus coding for this MP. In line with this assumption, it has been shown that production of the TMV MP is essential but insufficient by itself for complementation of foreign virus (comovirus) transport (Taliansky et al., 1992). In particular, the blockage or inhibition of the hybrid virus cell-to-cell spread in selective hosts may be due to the lack of a hypothetical host-specific or virus-specific factor(s) or/ and virus-induced host protein(s) in the hybrid-infected cells. When viruses with monocistronic transport genes were used for recombination, the foreign MPs in the resulting hybrid (and reassortant; Allison et al., 1988) viruses could efficiently substitute for one another in common hosts for both parental viruses (Nejidat et al., 1991; De Jong and Ahlquist, 1992; Hilf and Dawson, 1993; Richins et al., 1993; Mise and Ahlquist, 1995; Giesman-Cookmayer et al., 1994; Fenczik et al., 1995). Similar to the BSMV hybrid ND-MPT, the hybrid viruses of monocotadapted brome mosaic virus and dicot-adapted cowpea chlorotic mottle virus failed to support systemic infection and they also supported little or no cell-to-cell movement in the inoculated leaves of selective hosts of the parental viruses (Mise et al., 1993; Mise and Ahlquist, 1995). As shown by Fenczik et al. (1995), the replacement of the TMV MP gene by that of Odontoglossum ringspot tobamovirus allowed the hybrid to move systemically in or-

chids, whereas this hybrid virus was localized in the inoculated tobacco leaves (Hilf and Dawson, 1993). It should be noted that the hybrid was able to systemically infect N. benthamiana, a common systemic host for both viruses (Hilf and Dawson, 1993). BSMV and TMV produce necrotic lesions of distinct morphology on inoculated leaves of C. amaranticolor (Figs. 5A and 5B). It is noteworthy that the ND-MPT, where the TGB was replaced with the TMV 30K MP gene, produced necrotic lesions similar in size and morphology to those of TMV (Fig. 5C). Thus, the lesion phenotype was influenced by the foreign MP gene inserted into BSMV RNAb. This is in general agreement with the observations that the MPs may play a role in symptoms induction (Nejidat et al., 1991; Deom et al., 1994; Rao and Grantham, 1995; De Jong and Ahlquist, 1995). In contrast to wt BSMV, the BSMV hybrid containing the 30K TMV MP gene accumulated within the local lesions in extremely low amounts. This observation suggests that even negligible amounts of the ND-MPT recombinant can mediate sufficient cell-to-cell spread of the virus and subsequent host response to form the visible TMV-type lesions in C. amaranticolor. Taken together, our results show that the MP genes of distantly related plant viruses (hordei- and tobamoviruses) can substitute for each other in certain virus–host systems, and the MP genes may contribute to the lesion phenotype produced in a common host for both parental viruses.

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ACKNOWLEDGMENTS We are grateful to Dr. A. O. Jackson for providing the BSMV infectious cDND clones and Dr. A. A. Agranovsky for help in preparing the paper. This work was supported by INTAS Grant INTAS-93-0989.

REFERENCES Allison, R. F., Janda, M., and Ahlquist, P. (1988). Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus. J. Virol. 62, 3581–3588. Atabekov, J. G., and Taliansky, M. E. (1990). Expression of a plant viruscoded transport function by different viral genomes. Adv. Virus Res. 38, 201–247. Beck, D. L., Guilford, P. J., Voot, D. M., Andersen, M. T., and Forster, R. L. S. (1991). Triple block proteins of white clover mosaic potexvirus are required for transport. Virology 183, 695–702. Citovsky, V., and Zambryski, P. (1993). Transport of nucleic acids through membrane channels: Shaking through small holes. Annu. Rev. Microbiol. 47, 167–197. Dawson, W. O., and Hilf, M. E. (1992). Host-range determinations of plant viruses. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 43, 527–555. De Jong, W., and Ahlquist, P. (1992). A hybrid plant RNA virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection. Proc. Natl. Acad. Sci. USA 89, 6808–6812. De Jong, W., and Ahlquist, P. (1995). Host-specific alterations in viral RNA accumulation and infection spread in brome mosaic virus isolate with an expanded host range. J. Virol. 69, 1485–1492. Deom, C. M., Shaw, M., and Beachy, R. N. (1987). The 30-kilidalton

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gene product of tobacco mosaic virus potentiates virus movement. Science 327, 389–394. Deom, C. M., Lapidot, M., and Beachy, R. N. (1992). Plant virus movement proteins. Cell 69, 221–224. Deom, C. M., He, X. Z., Beachy, R. N., and Weissinger, A. K. (1994). Influence of heterologous tobamovirus movement protein and chimeric movement protein genes on cell-to-cell and long-distance movement. Virology 205, 198–209. Fenczik, C. A., Padgett, H. S., Holt, C. A., Casper, S. J., and Beachy, R. N. (1995). Mutational analysis of the movement protein of Odontoglossum ringspot virus to identify a host-range determinant. Mol. Plant–Microbe Interact. 8, 666–673. Fuentes, A. L., and Hamilton, R. I. (1991). Sunn-hemp mosaic virus facilitates cell-to-cell spread of southern bean mosaic virus in a nonpermissive host. Phytopathology 81, 1302–1305. Gal-on, A., Kaplan, I., Roossinck, M. J., and Palukaitis, P. (1994). The kinetics of infection of zucchini squash by cucumber mosaic virus indicate a function for RNA1 in virus movement. Virology 205, 280– 289. Giesman-Cookmayer, D., Lommel, S. A., and Deom, C. M. (1994). The TMV and RCNMV movement proteins are functionally homologous. In ‘‘Abstracts of the 13th Annual Meeting of American Society for Virology,’’ abstract W12-2, p. 145. Gilmer, D., Bouzoubaa, S., Hein, A., Guilley, H., Richards, K., and Jonard, G. (1992). Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3* proximal genes located on RNA2. Virology 189, 40– 47. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, M. E., Gait, M. J., and Karn, J. (1982). Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. USA 79, 5818–5822. Gustafson, G., and Armour, S. L. (1986). The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus. Nucleic Acids Res. 14, 3895–3909. Hamilton, R. I., and Dodds, J. A. (1970). Infection of barley by tobacco mosaic virus in single and mixed infection. Virology 42, 266–268. Hilf, M. E., and Dawson, W. O. (1993). The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement. Virology 193, 106–114. Ivanov, K. I., Ivanov, P. A., Timofeeva, E. K., Dorokhov, Yu, L., and Atabekov, J. G. (1994). The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA. FEBS Lett. 346, 213–220. Jackson, A. O., Petty, I. T. D., Jones, R. W., Edwards, M. C., and French, R. (1991). Analysis of barley stripe mosaic virus pathogenicity. Semin. Virol. 2, 107–119. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685. Lucas, W. J., and Gilbertson, R. L. (1994). Plasmodesmata in relation to viral movement within leaf tissues. Annu. Rev. Phytopathol. 32, 387–411. Malyshenko, S. I., Kondakova, O. A., Taliansky, M. E., and Atabekov, J. G. (1989). Plant virus transport function: Complementation by helper viruses is non-specific. J. Gen. Virol. 70, 2751–2757. Maule, A. (1991). Virus movement in infected plants. Crit. Rev. Plant Sci. 9, 457–473. Melcher, U. (1990). Similarities between putative transport proteins of plant viruses. J. Gen. Virol. 71, 1009–1018. Milne, R. G., and Lesemann, D.-E. (1984). Immunosorbent electron microscopy in plant virus studies. Methods Virol. 8, 85–101. Mise, K., and Ahlquist, P. (1995). Host-specificity restriction by bromovirus cell-to-cell movement protein occurs after initial cell-to-cell spread of infection in nonhost plants. Virology 206, 276–286.

Mise, K., Allison, R. F., Janda, M., and Ahlquist, P. (1993). Bromovirus movement protein genes play a crucial role in host specificity. J. Virol. 67, 2815–2823. Morozov, S. Yu., Dolja, V. V., and Atabekov, J. G. (1989). Probable reassortment of genomic elements among elongated RNA-containing plant viruses. J. Mol. Evol. 29, 52–62. Nejidat, A., Cellier, F., Holt, C. A., Gafny, R., Eggenberger, A. L., and Beachy, R. N. (1991). Transfer of the movement protein gene between two tobamoviruses: Influence on local lesion development. Virology 180, 318–326. Petty, I. T. D., and Jackson, A. O. (1990). Mutational analysis of barley stripe mosaic virus RNAb. Virology 179, 712–718. Petty, I. T. D., Hunter, B. G., and Jackson, A. O. (1988). A novel strategy for one step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus RNA. Gene 74, 423–432. Petty, I. T. D., Hunter, B. G., Wei, N., and Jackson, A. O. (1989). Infectious barley stripe mosaic virus RNA transcribed from full-length genomic cDNA clones. Virology 171, 342–349. Petty, I. T. D., French, R., Jones, R. W., and Jackson, A. O. (1990). Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. EMBO J. 9, 3453–3457. Petty, I. T. D., Donald, R. G. K., and Jackson, A. O. (1994). Multiple genetic determinants of barley stripe mosaic virus influence lesion phenotype on Chenopodium amaranticolor. Virology 198, 218–226. Rao, A. L. N., and Grantham, G. L. (1995). A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana. J. Virol. 69, 2689–2691. Richins, R. D., Donson, J., Lewandowsky, D. J., Ducasse, D. A., Dawson, W. O., and Shepherd, R. J. (1993). Temperature-dependent complementation of movement-deficient mutants of tobacco mosaic virus by gene I of peanut chlorotic streak caulimovirus. Phytopathology 83, 1349. Schneider, W. L., and Allison, R. F. (1993). Mutations that facilitate the substitution of the cowpea chlorotic mottle virus movement protein gene in brome mosaic virus. In ‘‘Abstracts of the 12th Annual Meeting of American Society for Virology,’’ abstract 34-1, p. A77. Solovyev, A. G., Novikov, V. K., Merits, A., Savenkov, E. I., Zelenina, D. A., Tyulkina, L. G., and Morozov, S. Yu. (1994). Genome characterization and taxonomy of Plantago asiatica mosaic potexvirus. J. Gen. Virol. 75, 259–267. Taliansky, M. E., Malyshenko, S. I., Kaplan, I. B., Kondakova, O. A., and Atabekov, J. G. (1992). Production of tobacco mosaic virus (TMV) transport protein in transgenic plants is essential but insufficient for complementing foreign virus transport: A need for the full-length TMV genome or some other TMV-encoded product. J. Gen. Virol. 73, 471– 474. Taliansky, M. E., de Jager, C. P., Wellink, J., van Lent, J. W. M., and Goldbach, R. W. (1993). Defective cell-to-cell movement of cowpea mosaic virus mutant N123 is efficiently complemented by sunn-hemp mosaic virus. J. Gen. Virol. 74, 1895–1901. Verwoerd, T. C., Dekker, B. M. M., and Hoekema, A. (1989). A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 17, 2106. Watanabe, Y., Morita, N., Nishiguchi, M., and Okada, Y. (1987). Attenuated strins of tobacco mosaic virus. Reduced synthesis of a viral protein with a cell-to-cell movement function. J. Mol. Biol. 194, 699– 704. Xiong, Z., Kim, K. H., Giesman-Cookmeyer, D., and Lommel, S. A. (1993). The roles of the red clover necrotic mosaic virus capsid and cell-tocell movement proteins in systemic infection. Virology 192, 27–32. Ziegler-Graff, V., Guilford, P. J., and Baulcombe, D. C. (1991). Tobacco rattle virus RNA-1 29K gene product potentiates viral movement and also affects symptom induction in tobacco. Virology 182, 145–155.

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