VIROLOGY
187, 499-507
(1992)
Effects of Terminal
Deletion Mutations on Function of the Movement of Tobacco Mosaic Virus
RON GAFNY,’ MOSHE LAPIDOT,2ANNE Department
BERNA, CURTIS A. HOLT,‘CARL
of Biology, Campus Box 1137, Washington Received September
17, 199 1; accepted
M. DEOM,4~~~
Universityz St. Louis, Missouri November
Protein
ROGER N. BEACHY’s
63 130
25, 799 1
A series of carboxy- and amino-terminal deletion mutations in the movement protein (MP) gene of tobacco mosaic virus (TMV) were ligated into a cloned TMV cDNA deleted for the endogenous MP gene. RNA transcripts were produced in vitro from clones carrying the various mutated MP genes. The effect of the deletion mutations on local and systemic movements of the infection was evaluated. Deletion of 9 or 33 amino acids from the carboxy terminus of the movement protein did not effect cell-to-cell movement as reflected by local lesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of 55 amino acids resulted in impaired MP that supported the formation of local lesions of 1 mm in diameter compared to lesions of 3-5 mm caused by the wild-type MP. Deletion of 74 amino acids (or more) from the carboxy terminus resulted in a protein that could not support virus movement. Modified viruses that contained repeated sequences in the 3’ region of the MP gene lost the repeated sequences during replication and reverted to the wild type. This was evidenced by the size of the MP produced and by sequence analysis of reverse-transcribed PCR-amplified products, following infection by the modified virus. MP deleted for as few as 3 amino acids at the amino terminus could not support virus movement thus indicating that the amino-terminal domain is critical for MP activity. 0 1992 Academic Press, Inc.
INTRODUCTION
containing the cell wall of TMV-infected tobacco plants and transgenic tobacco plants that express a gene encoding the MP (Deom et a/., 1987). lmmunogold cytochemistry showed that the movement protein is localized to the plasmodesmata of infected tobacco plants (Tomenius et al,, 1987) and transgenic plants producing the movement protein (Atkins et a/., 1991). The mechanisms by which the MP of TMV potentiates virus movement from cell-to-cell are not yet fully understood. Wolf et al., (1989) demonstrated that the protein has a direct effect on a plasmodesmatal function. The size exclusion limit of plasmodesmata in transgenic plants expressing the MP gene was larger than 9400 Da, whereas the size exclusion limit for control plants was 700 to 800 Da (Wolf et a/,, 1989). Citovsky et a/., (1990) demonstrated that the TMV MP binds single-stranded nucleic acid in vitro. They suggested that the protein functions by binding to the viral RNA and potentiates its transfer by keeping the RNA in a conformation compatible with cell-to-cell movement. Using transgenic plants expressing different TMV MPs truncated from the carboxy-terminal end, we demonstrated that the C-terminal 55 amino acids of the MP are not required for accumulation in a cell wall fraction, for plasmodesmatal modification, or for complementation of movement-defective TMV mutants (Berna et al., 1991). In the work reported here N-terminal and C-terminal deletions in the TMV MP gene were introduced
The successful development of infection by a plant virus requires the transfer of the virus from the initially infected cell to adjacent healthy cells. The cell-to-cell movement is not a passive diffusion process but requires an active transport function that is supplied by the virus (Atabekov and Dorokhov, 1984; Meshi et al., 1987; Deom et a/., 1987). The existence of a virus-encoded protein that potentiates cell-to-cell movement was first demonstrated for the 30-kDa protein of tobacco mosaic virus (TMV) (Leonard and Zaitlin, 1982; Meshi eta/., 1987; Deom et a/., 1987). The movement protein (MP) of TMV is a 30-kDa protein of 268 amino acids, translated from the I,-subgenomic RNA (Bruening et al., 1976). Cell fractionation experiments localized the MP to the fraction
’ Present address: The S. Tolkowski Laboratory, Department of Virology, A.R.O., The Volcani Center, Box 6, Bet Dagan 50250, Israel. ’ Present address: Division of Plant Biology-MRC7, Department of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA. 92037, U.S.A. 3 Present address: Universite Laval, Department de Biochimie, Ste-Foy. Quebec, Canada, Gl K 7P4. 4 Present address: Department of Plant Pathology, Miller plant science building, University of Georgia, Athens, GA. 30602 U.S.A. ’ To whom reprints requests should be addressed. 499
0042-6822192
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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GAFNY ET AL.
into an infectious clone of TMV. The effect of the different deletion mutations on local spread and long distance virus movement was studied. MATERIALS
AND METHODS
Plant lines /Vicotiana tabacum cvs. Xanthi and Xanthi NN were used as systemic and hypersensitive hosts, respectively. Transgenic tobacco plants expressing the MP gene of TMV strain Ul were Xanthi line 277 (Deom et al., 1987) and Xanthi NN line 2005 (Deom et a/., 1991). Growth and maintenance of plants were described by Deom et al., (1990). DNA manipulations, in vitro transcription and translation reactions, RNA extraction, and cDNA cloning DNA manipulations such as plasmid preparation, restriction, and ligation were essentially as described by Sambrook et al., (1989). DNA sequencing was done as described by Tabor and Richardson (1987). Transcription and translation reactions of amino-terminal-deleted MP genes were performed using T7 RNA polymerase and wheat germ extract according to the directions of the manufacturer (Promega). RNA was extracted from plants as described by Logemann et al., (1987). Reverse transcription and polymerase chain reactions were performed on total RNA preparations as described by Kawasaki (1990). The primers used for cDNA synthesis corresponded to TMV nucleotides 5876 to 5890 for first-strand synthesis and 4855 to 4874 for second-strand synthesis. (nucleotides were numbered according to Goelet et a/., 1982). Both primers were used for PCR amplification of the cDNA product. Construction MP genes
of infectious
clones carrying
deleted
A full-length cDNA clone of TMV Ul (Holt and Beachy, 1991) was modified by removing the sequences coding for either most of the movement protein (construct pU3/12AM-RV, Fig. 1) or both the movement protein and the coat protein (construct pU3/ AMAC, Fig. 1). RNA transcripts from pU3/12AM-RV and pU3/AMAC replicate only in initially infected cells in Xanthi and Xanthi NN and cannot move into adjacent cells unless a functional MP is cloned in the cDNA construct. The description of both clones will be provided elsewhere (Holt and Beachy, manuscript in preparation). The construction of C-terminal deletions in the MP gene was previously described (Berna et al., 1991).
The N-terminal deletions were constructed by site-directed mutagenesis using the in vitro mutagenesis system from Amersham. The MP gene used originated from plasmid pTM934 (Oliver eta/., 1986). The EcoRlBamHl fragment from pTM934 was cloned into the EcoRl -BamH 1 sites of pBluescript KS (Stratagene). ssDNA was prepared and used as a template for sitedirected mutagenesis. Synthetic oligonucleotides corresponding to 16-20 nucleotides from the 5’ and the 3’boundaries of each deletion were used to mutate the MP gene. The amino-terminal deletions were devised to retain the first two amino acids of the MP to preclude impairment in translation initiation. The cDNA of the mutated MPs was cut from pBluescript plasmids with EcoRl and BamH 1 restriction enzymes, made blunt by a fill-in reaction, and ligated into pU3/12AM-RV or pU3/12AMAC. The cloning was into the EcoRV site of pU3/12AM-RV or between the EcoRV and SnaBl sites of pU3/12AMAC (see Fig. 1). The DNA fragments cloned into pU3/12AMAC replaced the EcoRV-SnaBl fragment of this vector (Fig. 1). The cloning resulted in TMV infectious clones carrying truncated forms of the MP gene. In vitro transcription of infectious clones, inoculation, and detection of proteins in plant extracts Infectious transcripts of the different TMV clones were transcribed from the T7 promoter and mechanically inoculated as described before (Holt and Beachy, 1991). Disease development was followed after inoculation either with RNA directly from the in vitro transcription reaction or with virus extracted from local lesions that resulted from inoculation with transcript. Local lesion development was observed daily and lesion size was measured using a metric caliper. Cell wall fractions were isolated from leaf tissue as described by Deom et a/., (1990). The cell wall proteins were separated by SDS-PAGE in 12.5% gels (Laemmli, 1970) and blotted onto nitrocellulose. The MP and deletion mutants of the MP were detected with rabbit anti-TMV MP antibodies and ‘251-labeled donkey anti-rabbit antibodies (Amersham) (Harlow and Lane, 1988). The antibodies used were raised against synthetic peptides representing MP amino acids 6-26 (Berna et a/., 1991) or 205-223 (Deom et al., 1987) or against the MP produced in Escherichia co/i (R. Gafny, unpublished). RESULTS Characterization of carboxy-terminal deletion mutants of the MP in infectious TMV clones Deletion mutations at the carboxy-terminal end of the MP were previously expressed in transgenic to-
TMV MP TERMINAL
/ Sna Bl
pu3/l
/
ZAMAC
FIG. 1. Schematic diagram of the cloned cDNA of TMV RNA and modifications made to express mutant MP genes. (A) The full-length cDNA clone of Ul TMV RNA flanked by the T7 promoter and a unique Kpnl site was previously described (Holt and Beachy, 1991). The replicase represents TMV RNA nucleotides 68 to 4917, the MP (movement protein) nucleotides 4903 to 5709, and the CP (coat protein) nucleotides 57 12 to 6189. (B) pU3/12AM-RV was constructed by deleting most of the MP gene sequence (nucleotides 4923 to 5402) and inserting an EcoRV restriction site. pU3/12AMAC was constructed by deleting most of the coding region for the CP (nucleotides 5720 to 6170) from pU3/12AM-RV. OAS, origin of virus assembly. Arrows represent the subgenomic promoters for the MP and CP genes.
DELETION
501
MUTATIONS
Cloning of a truncated version of the MP into the EcoRV site of pU3/12AM-RV resulted in a virus that, upon infection, expresses the truncated MP gene, but retains the OAS and CP subgenomic promoter sequences. The viruses derived from the vector pU3/12AM-RV were designated AM. Viruses derived from pU3/ 12AMAC,which lacks both MP and CP (Fig. l), were designated AMAC. The C-terminal deletions and Nterminal deletions of the MP were designated CT and NT, respectively (Fig. 2). Thus the viruses carrying the truncated MP genes are designated according to the vector construct and the deletion mutation, e.g., AMCT2 or AMACNT3, etc. The infectivity of viruses derived from cDNA clones of pU3/12AM-RVcontaining the eight different C-terminal deletions (AMCTl-AMCT8) was assayed,as was a control construct carrying the intact MP gene (designated AMMP)as described in Fig. 2. RNA transcribed in vitro was inoculated to Xanthi and Xanthi NN. As summarized in Table 1, transcripts carrying the intact MP or carboxy-terminal deletions of up to 55 amino acids (AMMP-AMCT3) were infectious on wildtype tobacco plants. Transcripts carrying larger dele-
At380 I
II
A
B
0
MP
bacco plants (Berna et a/., 199 1). The same truncated MP genes were cloned into the TMV cDNAclone (Figs. 1 and 2) and the ability of the different MP mutants to support virus movement was studied. Within the 3’ region of the MP gene are two cis-acting sequences: the origin of assembly (OAS), which is necessary for virion assembly, and the promoter responsible for transcription of the coat protein (CP) subgenomic mRNA (Zimmern, 1977; Goelet et al., 1982). Deletion of the carboxy-terminal part of the MP may result in inability of the virus to produce coat protein or to be encapsidated. Coat protein production and encapsidation are necessary for long distance systemic spread of TMV but are not required for local cell-to-cell movement in the inoculated leaf (Siegel et a/., 1962; Takamatsu et al,, 1987; Dawson et a/., 1988; Saito et a/., 1990). To avoid the problems associated with the inability to produce virions, pU3/12AM-RV (Fig. 1) was used as the expression vector for the carboxy-terminal deletions. pU3/12AM-RV is a modified clone of TMV lacking a functional MP gene and was constructed by deleting most of the coding sequence of the MP gene except for the 3’ region containing the origin of assembly and the CP subgenomic promoter. A schematic representation of pU3/12AM-RV is shown in Fig. 1.
ACIDS
C
CT1
259-268
CT2
234-268
CT3
214-268
CT4
196-268
CT5
177-268
CT6
161-268
CT7
131-268
CT8
108-268
NT1
A
3-5
NT2
A
3-8
NT3
P
3-20
NT4
A
3-35
NT5
3-98
NT6
3-l
NT7
3-148
NT8 NT9
18
j-174 s=Tx
3-214
FIG. 2. Deletion mutants in the movement protein of TMV. The conserved domains of tobamoviruse movement proteins, regions I and II, and the acidic (A and C) and basic (B) regions (Saito er al., 1988) are indicated in the schematic representation of the MP gene. The deletion mutations of the carboxy terminus are labeled CT. The deletion mutation in the amino terminus are labeled NT. The aminoterminal deletions were constructed to maintain the first two amino acids residues; the extention of each deletion is indicated bythe thin lines.
502
GAFNY ET AL. TABLE 1 CHARACTERIZATIONOF MP DELETION MUTANTS IN TMV
Construct’
Amino acids deletedb
Infectivitp on tobaccod
Infectivity on MP+ tobacco”
MP CT1 CT2 CT3 CT4 CT5 CT6 CT7 CT8
0 9 33 55 74 94 111 132 161
+ + + + -
+ + + + + + + + +
NT1 NT2 NT3 NT4 NT5 NT6 NT7 NT8 NT9
3 6 19 33 96 116 146 172 212
-
+ + + + + + + + +
a CT, C-terminal deletions; NT, N-terminal deletions. b For the NT constructs the first and second amino acids were left intact. ’ Infectivity was determined by the appearance of local lesions on Xanthi NN and by symptom appearance on upper leaves of Xanthi plants. d Iv. tabacum cultivars Xanthi and Xanthi NN. eTransgenic plant lines 277 and 2005 which express the wildtype gene MP.
tions did not give detectable infection on wild-type tobacco plants (AMCT4to AMCT8).Systemic spread, or the lack of spread, was verified by inoculating the local lesion host Xanthi NN and transgenic plant line 2005 with homogenates of tissue taken from the upper leaf of a Xanthi tobacco plant inoculated with the different mutants. Transcripts of each of the AMCTsconstructs were infectious when inoculated to transgenic plants expressing the intact MP (plant lines 277 and 2005, Xanthi and Xanthi NN, respectively). The infectivity of transcripts from clones AMCT4-AMCT8 on MP-expressing plants indicates that the failure to infect wildtype tobacco plants is the result of a nonfunctional MP mutant. The rates of virus movement supported by the intact MP and the CTl-CT3 deletion mutants were compared by measuring the rate of local lesion expansion. It is assumed that the appearance and rate of lesion expansion on an infected Xanthi NN plant are a direct reflection of virus movement as is the case in TMV-infected N. glutinosa (Rappaport and Wildman, 1957). Xanthi NN plants were inoculated with the different
AMCT viruses with AMMP inoculated on opposite half leaves. The results presented in Fig. 3A suggest that viruses that express MPs lacking 9, 33, or 55 amino acids support virus movement to the same degree as wild-type MP when expressed in pU3/12AM-RV. To detect the wild-type MP expressed from the virus AMMP and the deleted MP expressed from the virus AMCT3, cell wall proteins were extracted from infected plants as described by Deom et a/., (1990). A protein corresponding to the electrophoretic mobility of the wild-type MP was detected in plants infected with AMCT3(Fig. 3B), as well as in those infected with AMMP. The wild-type size protein could be detected as early as 96 hr after inoculation with RNA produced in in vitro transcription reactions. We did not detect a protein corresponding to the expected size of the 55 amino acid deletion (data not shown). The same result was observed with AMCTl and AMCT2 (data not shown). The CT3 MP gene contains sequences that are repeated in pU3/12AM-RV; thus, the AMCT3virus contains a repeat of 139 nucleotides (nucleotides 5402 to 5541). The repeat is, however, interrupted by 16 nucleotides derived from the EcoRl linker containing a translational termination codon introduced during the construction of the deletion (Berna et a/., 1991). The synthesis of MP with the electrophoretic mobilityequivalent of wild-type MP by AMCT3 may result from deletion of the repeated sequences thereby reconstructing a wild-type MP gene. To determine if this is the case, the MP region was cloned from plants infected with AMCT3. Xanthi plants inoculated with transcripts of AMCT3were collected 96 hr after inoculation and total RNA was extracted. The RNA was subjected to reverse transcription and polymerase chain reaction using primers complementary to sequences from outside the repeated sequence. The resulting DNA fragment was cloned into pUCl9 and sequenced. The fragment contained only the sequence of wild-type TMV MP, indicating that there was a precise deletion of the repeated sequences. To overcome the problem of sequence deletion, we tested the activity of mutants CT1 to CT4 in a second TMV vector, pU3/12AMAC (Fig. 1). This vector was constructed from pU3/12AM-RV by deletion of most of the coat protein gene and insertion of a SnaBl restriction site as described under Materials and Methods. In this context the viruses carrying the deleted MP genes do not contain repeated sequences between the 3’end of the MP gene and the vector. The virus resulting from cloning of a functional movement protein gene into pU3/12AMAC would be expected to replicate and move from cell-to-cell in the inoculated leaf, but would not move systemically due to the absence of the coat
TMV MP TERMINAL
3
DELETION
-
AMCTl
__t_
AMCT2
-
AMCT3
503
MUTATIONS
2
0
1
2 DAYS
3 AFTER
4
5
6
7
INFECTION
B
18.4--w
FIG. 3. (A) Time course of necrotic lesion development on N. fabacum cv. Xanthi NN infected by AMMP, AMCTl , AMCT2, and AMCT3. Each virus was inoculated on half leaves.; opposite half leaves were inoculated with AMMP. Twenty to 30 lesions were measured for each treatment and each experiment was repeated three times. Since no differences were detected in AMMP lesion size among separate plants in a particular experiment, the results are presented as actual lesion size. Bars represent standard deviation. (B) Western immunoblot detection of MPs in the cell wall fraction. Lane 1, proteins from uninfected plants. Lane 2, proteins from AMMP-infected tissue. Lane 3, protein from AMCTB-infected tissue. Lane 4, protein from AMCT4-infectedtissue. Proteins were detected with antibodies against the MP and 1z51-labeled donkey anti-rabbit antibody as second antibody.
protein gene. The viruses con.strutted from ligating the MP deletions into pU3/12AMAC are designated by the prefix AMAC. Local lesions were observed after inoculation of N. tabacum cv. Xanthi NN with RNA transcripts derived from AMACMP or AMACCTl, AMACCT2 and AMACCT3. Inoculation with AMACCT4 (74 amino acids deleted) resulted in no infection of wild-type to-
bacco plants. AMACCT4 did infect transgenic plants expressing the Ul MP (line 2005), indicating that failure to infect wild-type tobacco plants is a result of a nonfunctional MP (Table 1). The virus carrying the 55 amino acid deletion (AMACCT3) gave smaller lesions than did viruses carrying the intact MP, the 9, or the 33 amino acid deletions. Lesion development was followed by com-
504
GAFNY ET AL. 6 1 5
4
--D-
AMACMP
__O_
AMACCTl
-
AMACCT2
-
AMACCTB
0
12
3 DAYS
4
5
AFTER
6
T
T
7
8
9
INFECTION
B
lesion development on N. tabacum cv. Xanthi NN infected by AMACMP, AMACCTl , AMACCT2, and on half leaves opposite to AMACMP as described in the legend for Fig. 3. (B) Western immunoblot detection of MPs in the cell wall fraction as described in the legend for Fig. 3. Lane 1, proteins from uninfected plants. Lane 2, proteins from AMACMP-infected tissue. Lane 3, protein from AMACCTB-infectedtissue. Lane 4, protein from AMACCT4-infectedtissue. FIG. 4. (A) Time course of necrotic
AMACCT3. Each RNA transcript was inoculated
paring
lesion
sizes on half leaves
inoculated
with
AMACMP (as control) and those produced by AMAC carrying the deletion mutant on opposite half leaves. Twenty to 30 lesions were measured per half leaf. The results presented in Fig. 4A show that there was a l-day delay in the appearance of lesions induced by AMACCT3 compared with wild-type or with AMACCTl and AMACCT2 mutants. The lesions produced by AMACCT3 stopped expanding after reaching a size of 1 mm, while lesions produced by the other viruses
reached 3-5 mm in diameter after a 7-day period (Fig. 4A). The CT3 MP could be detected in cell wall extracts from plants infected with AMACCT3 RNA and was of the expected electrophoretic mobility for the 55 amino acid deletion (Fig. 4B). To confirm that the small lesion phenotype of AMACCT3 is a result of impairment in the mutated movement protein rather then deficiency in replication, viral RNA was inoculated to Xanthi NN transgenic plants expressing the wild-type MP, i.e., plant line
TMV MP TERMINAL 1 43
+
35
-0
29
-w
2345676
9
IO
DELETION
505
MUTATIONS
tact MP (plant line 2005). These results indicate that the failure to establish infection on wild-type plants was due to the impairment of the MP. The results demonstrate that deletion of as few as three amino acids (amino acids 3 to 5) from the amino terminus of the MP results in a nonfunctional protein.
20 + I a.4+ I5
DISCUSSION -e
FIG. 5. In vitro translation of TMV MP genes deleted at the amino terminus, Lane 1, no RNA added to the translation reaction; lane 2, MP; lane 3, NT1 ; lane 4, NT2; lane 5, NT3; lane 6, NT4; lane 7, NT5; lane 8 NT6; lane 9, NT7; lane 10, NT8. The cloned cDNA in pBluescript KS were transcribed in vitro byT7 RNA polymerase. The RNA was used for in vitro translation reactions with the wheat germ system and 3H-labeled leucine. The translation products were separated on a 12.5% acrylamide gel, and 3H-labeled proteins were detected by fluorography. Transcription and translation reactions were as described by the manufacturer (Promega).
2005. Lesions produced by AMACCT3on plant line 2005 were equal in size to those produced by other AMAC constructs, indicating that lesion size caused by AMACCT3was the result of the MP itself (data not shown). Characterization of amino-terminal mutants of the MP
deletion
The MP cDNA was subjected to deletion from the amino terminus using site-directed mutagenesis. Each deletion was designed to preserve the first 2 amino acids of the MP to avoid affects on initiation of translation. Nine clones were generated that had deletions of between 3 and 212 amino acids (Fig. 2). To confirm that the deleted MP genes could direct the synthesis of the expected proteins, the cloned cDNAs were subjected to in vitro transcription and translation analysis using T7 RNA polymerase and a wheat germ translation system. In each case a translation product of the expected size corresponding to each particular mutant ORF was detected (Fig. 5). The deleted MP genes were cloned into the EcoRV site of pU3/12AM-RV as described above for the carboxy-terminal deletions (Fig. 1). The clones carrying the deleted genes were transcribed in vitro from the T7 promoter and transcripts were inoculated to tobacco plants as above. The results of these experiments are presented in Table 1. None of the mutants produced symptoms when inoculated to wild-type tobacco plants, although each one induced local lesions following inoculation to transgenic plants expressing the in-
Analysis of carboxy-terminal deletion mutants of the TMV MP in transgenic plants identified a stretch of 19 amino acids (amino acids 195 to 213) that are important for protein stability and/or localization of the protein to the plant cell walls, and for its activity (Berna et al., 1991). In the current study we extended the analysis of the MP by determining the effects of amino- and carboxy-terminal deletion mutants of the protein on its function during virus replication. Protein function was evaluated by expressing the mutated protein as a component of a modified strain of TMV. Deletion of 9 or 33 amino acids from the carboxy terminus of the MP resulted in a protein that could support virus movement. These deletions did not affect the rate of local spread of infection as deduced from the rate of expansion and final size of local lesions. We concluded from these results that the carboxy terminus of the MP is dispensable for cell-to-cell spread in inoculated leaves of Xanthi NN plants. Our experiments did not determine whether this region of the MP is necessary for long distance systemic movement or for infection of hosts other than tobacco. It is reasonable to assume, however, that the carboxy-terminal 33 amino acids are required for some function of the MP since it has been maintained as an open reading frame in the tobamoviruses characterized to date (Saito eta/,, 1988). This is, however, a highly divergent region in the MP in the tobamoviruses (Melcher, 1990). A MP with a deletion of 55 amino acids (CT3) supported virus movement but resulted in a small lesion phenotype. Complementation of the small lesion phenotype on transgenic plants expressing the wild-type MP confirmed that the small lesion resulted from impairment of the CT3 MP and not other limitations, e.g., an effect on virus replication. The activity of the carboxy-terminal mutations in the MP in infectious TMV transcripts is in agreement with the observation reported for the deletions expressed in transgenic plants (Berna et a/., 1991). In the study by Berna et a/., (1991) the effect of a particular mutation on lesion size or rate of virus movement was difficult to evaluate because of variations in the level of expression between different plant lines. Expression of the wild-type MP gene at unequal levels in independently transformed tobacco lines resulted in different lesion sizes (Deom et a/.,
506
GAFNY ET AL.
1991). Expression of the mutated MP gene from the endogenous movement protein subgenomic promoter allowed the direct evaluation of particular mutations on virus movement during virus infection. Citovskyetal., (1990) reported that the MP of TMV is a single-strand nucleic acid binding protein. Deletion analysis, based upon in v&o binding tests, mapped the binding site to amino acid residues 65-86 (Citovsky et al., 1990). Amino acid residues 203 to 251 comprise a domain that has a high proportion of basic amino acids (Saito et al., 1988). Citovsky et a/., (1990) did not identify this region as the nucleic acid binding site, although it might contribute to RNA binding in viva due to its highly positive charge. The reduced virus movement supported by the CT3 mutant might be the consequence of deleting 1 1 of the 15 basic amino acids residues in this domain. Our experiments with the “infectious” cDNA clone of TMV to assess the role of the carboxy-terminal region of the MP were complicated by the fact that the origin for virus assembly and the subgenomic promoter for the coat protein gene both overlap the coding sequence for the MP gene. We attempted to circumvent this problem by using a TMV vector that contained these sequences independent of the MP gene tested (pU3/12AM-RV). Unfortunately, the presence of repeated sequences between some of the MP deletion mutants and the vector resulted in deletion of these sequences during RNA replication and the reconstruction of the wild-type MP gene. Sequence deletion was first suspected when the MP produced in plants infected with AMCTSwas observed to have the same mobility in SDS-PAGE as wild-type MP. Reverse transcription, PCR cloning, and sequence analysis established that there was reversion to the wild-type sequence. This observation is similar to that reported by Lehto and Dawson (1990). In their experiment a TMV virus carrying a second copy of the MP gene (including a repeat of 8 1 1 nucleotides) reverted to wild-type virus. The deletion process and reconstruction of wild-type sequence of AMCT3(a repeat of 139 nucleotides) apparently occurred more rapidly than the deletion of 81 1 nucleotides observed by Lehto and Dawson (1990). The wild-type MP was detected in inoculated leaves 96 hr after infection by AMCT3 (Fig. 3B), while the virus carrying two copies of the MP gene was stable for more then 10 days (Lehto and Dawson, 1990). The rapid reversion of AMCT3 may reflect the impaired activity of the CT3 mutant, resulting in a wild-type virus population that dominates cell-to-cell movement. The analysis of amino-terminal deletions in the MP gene revealed that a deletion of three amino acids (residues 3-5; the smallest deletion tested) resulted in a nonfunctional protein. It is assumed that such a small
deletion at the amino terminus did not result in a gross structural change, and that the effect is directly on an important determinant for protein stability or activity. The amino terminus of the MP apparently does not function as a cleavable signal peptide (C. M. Deom and R. Gafny, unpublished). The analysis of the movement protein of TMV and other viruses will require genetic, cellular, and biochemical studies. Those described here represent an important first step in a future indepth study of the TMV MP.
ACKNOWLEDGMENTS We are grateful to M. Dyer and S. Leitner for growth and maintenance of plant materials. We also thank colleagues at the Monsanto Company for supplying oligonucleotides for mutagenesis during the early phase of this work. This work was supported by grants from the National Science Foundation to R.N.B. (DMB8717012), the Monsanto Company to R.N.B., and the Center for Plant Science and Biotechnology founded by the Missouri Centers for Advanced Technology. R.G. and M.L. were supported by BARD Fellowships.
REFERENCES ATABEKOV, J. G., and DOROKHOV, Y. L. (1984). Plant virus-specific transport function and resistance of plants to viruses. Adv. virus Res. 29,313-364. ATKINS, D., HULL, R., WELLS, B., ROBERTS,K., MOORE, P., and BEACHY, R. N. (1991). The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata. J. Gen. Viral. 72, 209-21 1. BERNA, A., GAFNY, R., WOLF, S., LUCAS, W. J., HOLT, C. A., and BEACHY, R. N. (1991). The TMV movement protein: Role of the C-terminal 73 amino acids in subcellular localization and function. Virology 182, 682-689. BRUENING, G., BEACHY, R. N., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in viva translation of ribonucleic acids of cowpea strain of tobacco mosaic virus. Virology 71, 498-517. CITOVSKY,V., KNORR,D., SCHUSTER,G., and ZAMBRYSKI,P. (1990). The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637-647. DAWSON, W. O., BUBRICK, P., and GRANTHAM, G. L. (1988). Modification of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomology. Phytopathology 78, 783789. DEOM, C. M., OLIVER, M. J., and BEACHY, R. N. (1987). The 30-Kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science 237, 384-389. DEOM, C. M., SCHUBERT, K. R., WOLF, S., HOLT, C. A., LUCAS, W. J., and BEACHY, R. N. (1990). Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants. froc. Nat/. Acad. Sci. USA 87, 3284-3288. DEOM, C. M., WOLF, S., HOLT, C. A., LUCAS, W. J., and BEACHY, R. N. (1991). Altered function of the tobacco mosaic virus movement protein in a hypersensitive host. virology 180, 251-256. 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. hoc. Nat/. Acad. Sci. USA 79, 58 18-5822. HARLOW, E., and LANE, D. (1988). “Antibodies: A Laboratory Manual.” Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. HOLT, C. A., and BEACHY, R. N. (1991). ln viva complementation of
TMV MP TERMINAL infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. virology 181, 109-l 17. KAWASAKI,E. S. (1990). cDNA cloning using degenerate primers. ln “PCR Protocols” (M. A. Innis, D. H. Gelfand, J. J. Sninsky, andT. J. White, Eds.), pp. 21-27. Academic Press, San Diego. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LEHTO, K., and DAWSON,W. 0. (1990). Replication, stability, and gene expression of tobacco mosaic virus mutants with a second 30K ORF. Virology 175, 30-40. LEONARD, D. A., and ZAITLIN, M. (1982). A temperature-sensitive strain of tobacco mosaic virus defective in cell to cell movement generates an altered virus-coded protein. virology 117, 416-424. LOGEMANN, J., SCHELL, J., and WILLMITZER, L. (1987). Improved method for isolation of RNA from plant tissue. Anal. Biochem. 163, 16-20. MELCHER, U. (1990). Similarities between putative transport proteins of plant viruses. /. Gen. Viral. 71, 1009-l 018. MESHI, T., WATANABE, Y., SAITO, T., SUGIMOTO, A., MAEDA, T., and OKADA, Y. (1987). Function of the 30kD protein of tobacco mosaic virus: Involvement in cell-to-cell movement and dispensibility for replication. EMBO J. 6, 2557-2563. OLIVER, M. J., DEOM, C. M., and BEACHY, R. N. (1986). In vitro transcription and translation of cloned cDNAs encoding the 30-kDa protein gene of TMV. Virology 155, 277-283. RAPPAPORT,I., and WILDMAN, S. G. (1957). A kinetic study of local
DELETION
MUTATIONS
507
lesion growth on Nicotiana glutinosa resulting from tobacco mosaic virus infection. Virology 4, 265-274. SAMBROOK. J., FRITSCH, E. F., and MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. SAITO, T., IMAI, Y., MESHI, T., and OKADA, Y. (1988). Interviral homologies of the 30K protein of tobamoviruses. Virology 167, 653-656. SAITO, T., YAMANAKA, K., and OKADA, Y. (1990). Long-distance movement and viral assembly of TMV mutants. Virology 176, 329-336. SEIGEL, A., ZAITLIN, M., and SEHGAL, 0. P. (1962). The isolation of defective tobacco mosaic virus strains. Proc. Nat/. Acad. Sci. USA 48, 1845-1851. TABOR, S., and RICHARDSON,C. C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Nat/. Acad. Sci. USA 84, 4767-477 1. TAKAMATSU, N., ISHIKAWA, M., MESHI, T., and OKADA, Y. (1987). Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO /. 6, 307-31 1. TOMENIUS, K., CLAPHAM, D., and MESHI, T. (1987). Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160, 363-37 1. WOLF, S., DEOM, C. M., BEACHY, R. N., and LUCAS, W. J. (1989). Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246, 377-379. ZIMMERN, D. (1977). The nucleotide sequence at the origin for assembly on tobacco mosaic virus RNA. Cell 11, 463-482.
187, 499-507
(1992)
Effects of Terminal
Deletion Mutations on Function of the Movement of Tobacco Mosaic Virus
RON GAFNY,’ MOSHE LAPIDOT,2ANNE Department
BERNA, CURTIS A. HOLT,‘CARL
of Biology, Campus Box 1137, Washington Received September
17, 199 1; accepted
M. DEOM,4~~~
Universityz St. Louis, Missouri November
Protein
ROGER N. BEACHY’s
63 130
25, 799 1
A series of carboxy- and amino-terminal deletion mutations in the movement protein (MP) gene of tobacco mosaic virus (TMV) were ligated into a cloned TMV cDNA deleted for the endogenous MP gene. RNA transcripts were produced in vitro from clones carrying the various mutated MP genes. The effect of the deletion mutations on local and systemic movements of the infection was evaluated. Deletion of 9 or 33 amino acids from the carboxy terminus of the movement protein did not effect cell-to-cell movement as reflected by local lesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of 55 amino acids resulted in impaired MP that supported the formation of local lesions of 1 mm in diameter compared to lesions of 3-5 mm caused by the wild-type MP. Deletion of 74 amino acids (or more) from the carboxy terminus resulted in a protein that could not support virus movement. Modified viruses that contained repeated sequences in the 3’ region of the MP gene lost the repeated sequences during replication and reverted to the wild type. This was evidenced by the size of the MP produced and by sequence analysis of reverse-transcribed PCR-amplified products, following infection by the modified virus. MP deleted for as few as 3 amino acids at the amino terminus could not support virus movement thus indicating that the amino-terminal domain is critical for MP activity. 0 1992 Academic Press, Inc.
INTRODUCTION
containing the cell wall of TMV-infected tobacco plants and transgenic tobacco plants that express a gene encoding the MP (Deom et a/., 1987). lmmunogold cytochemistry showed that the movement protein is localized to the plasmodesmata of infected tobacco plants (Tomenius et al,, 1987) and transgenic plants producing the movement protein (Atkins et a/., 1991). The mechanisms by which the MP of TMV potentiates virus movement from cell-to-cell are not yet fully understood. Wolf et al., (1989) demonstrated that the protein has a direct effect on a plasmodesmatal function. The size exclusion limit of plasmodesmata in transgenic plants expressing the MP gene was larger than 9400 Da, whereas the size exclusion limit for control plants was 700 to 800 Da (Wolf et a/,, 1989). Citovsky et a/., (1990) demonstrated that the TMV MP binds single-stranded nucleic acid in vitro. They suggested that the protein functions by binding to the viral RNA and potentiates its transfer by keeping the RNA in a conformation compatible with cell-to-cell movement. Using transgenic plants expressing different TMV MPs truncated from the carboxy-terminal end, we demonstrated that the C-terminal 55 amino acids of the MP are not required for accumulation in a cell wall fraction, for plasmodesmatal modification, or for complementation of movement-defective TMV mutants (Berna et al., 1991). In the work reported here N-terminal and C-terminal deletions in the TMV MP gene were introduced
The successful development of infection by a plant virus requires the transfer of the virus from the initially infected cell to adjacent healthy cells. The cell-to-cell movement is not a passive diffusion process but requires an active transport function that is supplied by the virus (Atabekov and Dorokhov, 1984; Meshi et al., 1987; Deom et a/., 1987). The existence of a virus-encoded protein that potentiates cell-to-cell movement was first demonstrated for the 30-kDa protein of tobacco mosaic virus (TMV) (Leonard and Zaitlin, 1982; Meshi eta/., 1987; Deom et a/., 1987). The movement protein (MP) of TMV is a 30-kDa protein of 268 amino acids, translated from the I,-subgenomic RNA (Bruening et al., 1976). Cell fractionation experiments localized the MP to the fraction
’ Present address: The S. Tolkowski Laboratory, Department of Virology, A.R.O., The Volcani Center, Box 6, Bet Dagan 50250, Israel. ’ Present address: Division of Plant Biology-MRC7, Department of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA. 92037, U.S.A. 3 Present address: Universite Laval, Department de Biochimie, Ste-Foy. Quebec, Canada, Gl K 7P4. 4 Present address: Department of Plant Pathology, Miller plant science building, University of Georgia, Athens, GA. 30602 U.S.A. ’ To whom reprints requests should be addressed. 499
0042-6822192
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
500
GAFNY ET AL.
into an infectious clone of TMV. The effect of the different deletion mutations on local spread and long distance virus movement was studied. MATERIALS
AND METHODS
Plant lines /Vicotiana tabacum cvs. Xanthi and Xanthi NN were used as systemic and hypersensitive hosts, respectively. Transgenic tobacco plants expressing the MP gene of TMV strain Ul were Xanthi line 277 (Deom et al., 1987) and Xanthi NN line 2005 (Deom et a/., 1991). Growth and maintenance of plants were described by Deom et al., (1990). DNA manipulations, in vitro transcription and translation reactions, RNA extraction, and cDNA cloning DNA manipulations such as plasmid preparation, restriction, and ligation were essentially as described by Sambrook et al., (1989). DNA sequencing was done as described by Tabor and Richardson (1987). Transcription and translation reactions of amino-terminal-deleted MP genes were performed using T7 RNA polymerase and wheat germ extract according to the directions of the manufacturer (Promega). RNA was extracted from plants as described by Logemann et al., (1987). Reverse transcription and polymerase chain reactions were performed on total RNA preparations as described by Kawasaki (1990). The primers used for cDNA synthesis corresponded to TMV nucleotides 5876 to 5890 for first-strand synthesis and 4855 to 4874 for second-strand synthesis. (nucleotides were numbered according to Goelet et a/., 1982). Both primers were used for PCR amplification of the cDNA product. Construction MP genes
of infectious
clones carrying
deleted
A full-length cDNA clone of TMV Ul (Holt and Beachy, 1991) was modified by removing the sequences coding for either most of the movement protein (construct pU3/12AM-RV, Fig. 1) or both the movement protein and the coat protein (construct pU3/ AMAC, Fig. 1). RNA transcripts from pU3/12AM-RV and pU3/AMAC replicate only in initially infected cells in Xanthi and Xanthi NN and cannot move into adjacent cells unless a functional MP is cloned in the cDNA construct. The description of both clones will be provided elsewhere (Holt and Beachy, manuscript in preparation). The construction of C-terminal deletions in the MP gene was previously described (Berna et al., 1991).
The N-terminal deletions were constructed by site-directed mutagenesis using the in vitro mutagenesis system from Amersham. The MP gene used originated from plasmid pTM934 (Oliver eta/., 1986). The EcoRlBamHl fragment from pTM934 was cloned into the EcoRl -BamH 1 sites of pBluescript KS (Stratagene). ssDNA was prepared and used as a template for sitedirected mutagenesis. Synthetic oligonucleotides corresponding to 16-20 nucleotides from the 5’ and the 3’boundaries of each deletion were used to mutate the MP gene. The amino-terminal deletions were devised to retain the first two amino acids of the MP to preclude impairment in translation initiation. The cDNA of the mutated MPs was cut from pBluescript plasmids with EcoRl and BamH 1 restriction enzymes, made blunt by a fill-in reaction, and ligated into pU3/12AM-RV or pU3/12AMAC. The cloning was into the EcoRV site of pU3/12AM-RV or between the EcoRV and SnaBl sites of pU3/12AMAC (see Fig. 1). The DNA fragments cloned into pU3/12AMAC replaced the EcoRV-SnaBl fragment of this vector (Fig. 1). The cloning resulted in TMV infectious clones carrying truncated forms of the MP gene. In vitro transcription of infectious clones, inoculation, and detection of proteins in plant extracts Infectious transcripts of the different TMV clones were transcribed from the T7 promoter and mechanically inoculated as described before (Holt and Beachy, 1991). Disease development was followed after inoculation either with RNA directly from the in vitro transcription reaction or with virus extracted from local lesions that resulted from inoculation with transcript. Local lesion development was observed daily and lesion size was measured using a metric caliper. Cell wall fractions were isolated from leaf tissue as described by Deom et a/., (1990). The cell wall proteins were separated by SDS-PAGE in 12.5% gels (Laemmli, 1970) and blotted onto nitrocellulose. The MP and deletion mutants of the MP were detected with rabbit anti-TMV MP antibodies and ‘251-labeled donkey anti-rabbit antibodies (Amersham) (Harlow and Lane, 1988). The antibodies used were raised against synthetic peptides representing MP amino acids 6-26 (Berna et a/., 1991) or 205-223 (Deom et al., 1987) or against the MP produced in Escherichia co/i (R. Gafny, unpublished). RESULTS Characterization of carboxy-terminal deletion mutants of the MP in infectious TMV clones Deletion mutations at the carboxy-terminal end of the MP were previously expressed in transgenic to-
TMV MP TERMINAL
/ Sna Bl
pu3/l
/
ZAMAC
FIG. 1. Schematic diagram of the cloned cDNA of TMV RNA and modifications made to express mutant MP genes. (A) The full-length cDNA clone of Ul TMV RNA flanked by the T7 promoter and a unique Kpnl site was previously described (Holt and Beachy, 1991). The replicase represents TMV RNA nucleotides 68 to 4917, the MP (movement protein) nucleotides 4903 to 5709, and the CP (coat protein) nucleotides 57 12 to 6189. (B) pU3/12AM-RV was constructed by deleting most of the MP gene sequence (nucleotides 4923 to 5402) and inserting an EcoRV restriction site. pU3/12AMAC was constructed by deleting most of the coding region for the CP (nucleotides 5720 to 6170) from pU3/12AM-RV. OAS, origin of virus assembly. Arrows represent the subgenomic promoters for the MP and CP genes.
DELETION
501
MUTATIONS
Cloning of a truncated version of the MP into the EcoRV site of pU3/12AM-RV resulted in a virus that, upon infection, expresses the truncated MP gene, but retains the OAS and CP subgenomic promoter sequences. The viruses derived from the vector pU3/12AM-RV were designated AM. Viruses derived from pU3/ 12AMAC,which lacks both MP and CP (Fig. l), were designated AMAC. The C-terminal deletions and Nterminal deletions of the MP were designated CT and NT, respectively (Fig. 2). Thus the viruses carrying the truncated MP genes are designated according to the vector construct and the deletion mutation, e.g., AMCT2 or AMACNT3, etc. The infectivity of viruses derived from cDNA clones of pU3/12AM-RVcontaining the eight different C-terminal deletions (AMCTl-AMCT8) was assayed,as was a control construct carrying the intact MP gene (designated AMMP)as described in Fig. 2. RNA transcribed in vitro was inoculated to Xanthi and Xanthi NN. As summarized in Table 1, transcripts carrying the intact MP or carboxy-terminal deletions of up to 55 amino acids (AMMP-AMCT3) were infectious on wildtype tobacco plants. Transcripts carrying larger dele-
At380 I
II
A
B
0
MP
bacco plants (Berna et a/., 199 1). The same truncated MP genes were cloned into the TMV cDNAclone (Figs. 1 and 2) and the ability of the different MP mutants to support virus movement was studied. Within the 3’ region of the MP gene are two cis-acting sequences: the origin of assembly (OAS), which is necessary for virion assembly, and the promoter responsible for transcription of the coat protein (CP) subgenomic mRNA (Zimmern, 1977; Goelet et al., 1982). Deletion of the carboxy-terminal part of the MP may result in inability of the virus to produce coat protein or to be encapsidated. Coat protein production and encapsidation are necessary for long distance systemic spread of TMV but are not required for local cell-to-cell movement in the inoculated leaf (Siegel et a/., 1962; Takamatsu et al,, 1987; Dawson et a/., 1988; Saito et a/., 1990). To avoid the problems associated with the inability to produce virions, pU3/12AM-RV (Fig. 1) was used as the expression vector for the carboxy-terminal deletions. pU3/12AM-RV is a modified clone of TMV lacking a functional MP gene and was constructed by deleting most of the coding sequence of the MP gene except for the 3’ region containing the origin of assembly and the CP subgenomic promoter. A schematic representation of pU3/12AM-RV is shown in Fig. 1.
ACIDS
C
CT1
259-268
CT2
234-268
CT3
214-268
CT4
196-268
CT5
177-268
CT6
161-268
CT7
131-268
CT8
108-268
NT1
A
3-5
NT2
A
3-8
NT3
P
3-20
NT4
A
3-35
NT5
3-98
NT6
3-l
NT7
3-148
NT8 NT9
18
j-174 s=Tx
3-214
FIG. 2. Deletion mutants in the movement protein of TMV. The conserved domains of tobamoviruse movement proteins, regions I and II, and the acidic (A and C) and basic (B) regions (Saito er al., 1988) are indicated in the schematic representation of the MP gene. The deletion mutations of the carboxy terminus are labeled CT. The deletion mutation in the amino terminus are labeled NT. The aminoterminal deletions were constructed to maintain the first two amino acids residues; the extention of each deletion is indicated bythe thin lines.
502
GAFNY ET AL. TABLE 1 CHARACTERIZATIONOF MP DELETION MUTANTS IN TMV
Construct’
Amino acids deletedb
Infectivitp on tobaccod
Infectivity on MP+ tobacco”
MP CT1 CT2 CT3 CT4 CT5 CT6 CT7 CT8
0 9 33 55 74 94 111 132 161
+ + + + -
+ + + + + + + + +
NT1 NT2 NT3 NT4 NT5 NT6 NT7 NT8 NT9
3 6 19 33 96 116 146 172 212
-
+ + + + + + + + +
a CT, C-terminal deletions; NT, N-terminal deletions. b For the NT constructs the first and second amino acids were left intact. ’ Infectivity was determined by the appearance of local lesions on Xanthi NN and by symptom appearance on upper leaves of Xanthi plants. d Iv. tabacum cultivars Xanthi and Xanthi NN. eTransgenic plant lines 277 and 2005 which express the wildtype gene MP.
tions did not give detectable infection on wild-type tobacco plants (AMCT4to AMCT8).Systemic spread, or the lack of spread, was verified by inoculating the local lesion host Xanthi NN and transgenic plant line 2005 with homogenates of tissue taken from the upper leaf of a Xanthi tobacco plant inoculated with the different mutants. Transcripts of each of the AMCTsconstructs were infectious when inoculated to transgenic plants expressing the intact MP (plant lines 277 and 2005, Xanthi and Xanthi NN, respectively). The infectivity of transcripts from clones AMCT4-AMCT8 on MP-expressing plants indicates that the failure to infect wildtype tobacco plants is the result of a nonfunctional MP mutant. The rates of virus movement supported by the intact MP and the CTl-CT3 deletion mutants were compared by measuring the rate of local lesion expansion. It is assumed that the appearance and rate of lesion expansion on an infected Xanthi NN plant are a direct reflection of virus movement as is the case in TMV-infected N. glutinosa (Rappaport and Wildman, 1957). Xanthi NN plants were inoculated with the different
AMCT viruses with AMMP inoculated on opposite half leaves. The results presented in Fig. 3A suggest that viruses that express MPs lacking 9, 33, or 55 amino acids support virus movement to the same degree as wild-type MP when expressed in pU3/12AM-RV. To detect the wild-type MP expressed from the virus AMMP and the deleted MP expressed from the virus AMCT3, cell wall proteins were extracted from infected plants as described by Deom et a/., (1990). A protein corresponding to the electrophoretic mobility of the wild-type MP was detected in plants infected with AMCT3(Fig. 3B), as well as in those infected with AMMP. The wild-type size protein could be detected as early as 96 hr after inoculation with RNA produced in in vitro transcription reactions. We did not detect a protein corresponding to the expected size of the 55 amino acid deletion (data not shown). The same result was observed with AMCTl and AMCT2 (data not shown). The CT3 MP gene contains sequences that are repeated in pU3/12AM-RV; thus, the AMCT3virus contains a repeat of 139 nucleotides (nucleotides 5402 to 5541). The repeat is, however, interrupted by 16 nucleotides derived from the EcoRl linker containing a translational termination codon introduced during the construction of the deletion (Berna et a/., 1991). The synthesis of MP with the electrophoretic mobilityequivalent of wild-type MP by AMCT3 may result from deletion of the repeated sequences thereby reconstructing a wild-type MP gene. To determine if this is the case, the MP region was cloned from plants infected with AMCT3. Xanthi plants inoculated with transcripts of AMCT3were collected 96 hr after inoculation and total RNA was extracted. The RNA was subjected to reverse transcription and polymerase chain reaction using primers complementary to sequences from outside the repeated sequence. The resulting DNA fragment was cloned into pUCl9 and sequenced. The fragment contained only the sequence of wild-type TMV MP, indicating that there was a precise deletion of the repeated sequences. To overcome the problem of sequence deletion, we tested the activity of mutants CT1 to CT4 in a second TMV vector, pU3/12AMAC (Fig. 1). This vector was constructed from pU3/12AM-RV by deletion of most of the coat protein gene and insertion of a SnaBl restriction site as described under Materials and Methods. In this context the viruses carrying the deleted MP genes do not contain repeated sequences between the 3’end of the MP gene and the vector. The virus resulting from cloning of a functional movement protein gene into pU3/12AMAC would be expected to replicate and move from cell-to-cell in the inoculated leaf, but would not move systemically due to the absence of the coat
TMV MP TERMINAL
3
DELETION
-
AMCTl
__t_
AMCT2
-
AMCT3
503
MUTATIONS
2
0
1
2 DAYS
3 AFTER
4
5
6
7
INFECTION
B
18.4--w
FIG. 3. (A) Time course of necrotic lesion development on N. fabacum cv. Xanthi NN infected by AMMP, AMCTl , AMCT2, and AMCT3. Each virus was inoculated on half leaves.; opposite half leaves were inoculated with AMMP. Twenty to 30 lesions were measured for each treatment and each experiment was repeated three times. Since no differences were detected in AMMP lesion size among separate plants in a particular experiment, the results are presented as actual lesion size. Bars represent standard deviation. (B) Western immunoblot detection of MPs in the cell wall fraction. Lane 1, proteins from uninfected plants. Lane 2, proteins from AMMP-infected tissue. Lane 3, protein from AMCTB-infected tissue. Lane 4, protein from AMCT4-infectedtissue. Proteins were detected with antibodies against the MP and 1z51-labeled donkey anti-rabbit antibody as second antibody.
protein gene. The viruses con.strutted from ligating the MP deletions into pU3/12AMAC are designated by the prefix AMAC. Local lesions were observed after inoculation of N. tabacum cv. Xanthi NN with RNA transcripts derived from AMACMP or AMACCTl, AMACCT2 and AMACCT3. Inoculation with AMACCT4 (74 amino acids deleted) resulted in no infection of wild-type to-
bacco plants. AMACCT4 did infect transgenic plants expressing the Ul MP (line 2005), indicating that failure to infect wild-type tobacco plants is a result of a nonfunctional MP (Table 1). The virus carrying the 55 amino acid deletion (AMACCT3) gave smaller lesions than did viruses carrying the intact MP, the 9, or the 33 amino acid deletions. Lesion development was followed by com-
504
GAFNY ET AL. 6 1 5
4
--D-
AMACMP
__O_
AMACCTl
-
AMACCT2
-
AMACCTB
0
12
3 DAYS
4
5
AFTER
6
T
T
7
8
9
INFECTION
B
lesion development on N. tabacum cv. Xanthi NN infected by AMACMP, AMACCTl , AMACCT2, and on half leaves opposite to AMACMP as described in the legend for Fig. 3. (B) Western immunoblot detection of MPs in the cell wall fraction as described in the legend for Fig. 3. Lane 1, proteins from uninfected plants. Lane 2, proteins from AMACMP-infected tissue. Lane 3, protein from AMACCTB-infectedtissue. Lane 4, protein from AMACCT4-infectedtissue. FIG. 4. (A) Time course of necrotic
AMACCT3. Each RNA transcript was inoculated
paring
lesion
sizes on half leaves
inoculated
with
AMACMP (as control) and those produced by AMAC carrying the deletion mutant on opposite half leaves. Twenty to 30 lesions were measured per half leaf. The results presented in Fig. 4A show that there was a l-day delay in the appearance of lesions induced by AMACCT3 compared with wild-type or with AMACCTl and AMACCT2 mutants. The lesions produced by AMACCT3 stopped expanding after reaching a size of 1 mm, while lesions produced by the other viruses
reached 3-5 mm in diameter after a 7-day period (Fig. 4A). The CT3 MP could be detected in cell wall extracts from plants infected with AMACCT3 RNA and was of the expected electrophoretic mobility for the 55 amino acid deletion (Fig. 4B). To confirm that the small lesion phenotype of AMACCT3 is a result of impairment in the mutated movement protein rather then deficiency in replication, viral RNA was inoculated to Xanthi NN transgenic plants expressing the wild-type MP, i.e., plant line
TMV MP TERMINAL 1 43
+
35
-0
29
-w
2345676
9
IO
DELETION
505
MUTATIONS
tact MP (plant line 2005). These results indicate that the failure to establish infection on wild-type plants was due to the impairment of the MP. The results demonstrate that deletion of as few as three amino acids (amino acids 3 to 5) from the amino terminus of the MP results in a nonfunctional protein.
20 + I a.4+ I5
DISCUSSION -e
FIG. 5. In vitro translation of TMV MP genes deleted at the amino terminus, Lane 1, no RNA added to the translation reaction; lane 2, MP; lane 3, NT1 ; lane 4, NT2; lane 5, NT3; lane 6, NT4; lane 7, NT5; lane 8 NT6; lane 9, NT7; lane 10, NT8. The cloned cDNA in pBluescript KS were transcribed in vitro byT7 RNA polymerase. The RNA was used for in vitro translation reactions with the wheat germ system and 3H-labeled leucine. The translation products were separated on a 12.5% acrylamide gel, and 3H-labeled proteins were detected by fluorography. Transcription and translation reactions were as described by the manufacturer (Promega).
2005. Lesions produced by AMACCT3on plant line 2005 were equal in size to those produced by other AMAC constructs, indicating that lesion size caused by AMACCT3was the result of the MP itself (data not shown). Characterization of amino-terminal mutants of the MP
deletion
The MP cDNA was subjected to deletion from the amino terminus using site-directed mutagenesis. Each deletion was designed to preserve the first 2 amino acids of the MP to avoid affects on initiation of translation. Nine clones were generated that had deletions of between 3 and 212 amino acids (Fig. 2). To confirm that the deleted MP genes could direct the synthesis of the expected proteins, the cloned cDNAs were subjected to in vitro transcription and translation analysis using T7 RNA polymerase and a wheat germ translation system. In each case a translation product of the expected size corresponding to each particular mutant ORF was detected (Fig. 5). The deleted MP genes were cloned into the EcoRV site of pU3/12AM-RV as described above for the carboxy-terminal deletions (Fig. 1). The clones carrying the deleted genes were transcribed in vitro from the T7 promoter and transcripts were inoculated to tobacco plants as above. The results of these experiments are presented in Table 1. None of the mutants produced symptoms when inoculated to wild-type tobacco plants, although each one induced local lesions following inoculation to transgenic plants expressing the in-
Analysis of carboxy-terminal deletion mutants of the TMV MP in transgenic plants identified a stretch of 19 amino acids (amino acids 195 to 213) that are important for protein stability and/or localization of the protein to the plant cell walls, and for its activity (Berna et al., 1991). In the current study we extended the analysis of the MP by determining the effects of amino- and carboxy-terminal deletion mutants of the protein on its function during virus replication. Protein function was evaluated by expressing the mutated protein as a component of a modified strain of TMV. Deletion of 9 or 33 amino acids from the carboxy terminus of the MP resulted in a protein that could support virus movement. These deletions did not affect the rate of local spread of infection as deduced from the rate of expansion and final size of local lesions. We concluded from these results that the carboxy terminus of the MP is dispensable for cell-to-cell spread in inoculated leaves of Xanthi NN plants. Our experiments did not determine whether this region of the MP is necessary for long distance systemic movement or for infection of hosts other than tobacco. It is reasonable to assume, however, that the carboxy-terminal 33 amino acids are required for some function of the MP since it has been maintained as an open reading frame in the tobamoviruses characterized to date (Saito eta/,, 1988). This is, however, a highly divergent region in the MP in the tobamoviruses (Melcher, 1990). A MP with a deletion of 55 amino acids (CT3) supported virus movement but resulted in a small lesion phenotype. Complementation of the small lesion phenotype on transgenic plants expressing the wild-type MP confirmed that the small lesion resulted from impairment of the CT3 MP and not other limitations, e.g., an effect on virus replication. The activity of the carboxy-terminal mutations in the MP in infectious TMV transcripts is in agreement with the observation reported for the deletions expressed in transgenic plants (Berna et a/., 1991). In the study by Berna et a/., (1991) the effect of a particular mutation on lesion size or rate of virus movement was difficult to evaluate because of variations in the level of expression between different plant lines. Expression of the wild-type MP gene at unequal levels in independently transformed tobacco lines resulted in different lesion sizes (Deom et a/.,
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1991). Expression of the mutated MP gene from the endogenous movement protein subgenomic promoter allowed the direct evaluation of particular mutations on virus movement during virus infection. Citovskyetal., (1990) reported that the MP of TMV is a single-strand nucleic acid binding protein. Deletion analysis, based upon in v&o binding tests, mapped the binding site to amino acid residues 65-86 (Citovsky et al., 1990). Amino acid residues 203 to 251 comprise a domain that has a high proportion of basic amino acids (Saito et al., 1988). Citovsky et a/., (1990) did not identify this region as the nucleic acid binding site, although it might contribute to RNA binding in viva due to its highly positive charge. The reduced virus movement supported by the CT3 mutant might be the consequence of deleting 1 1 of the 15 basic amino acids residues in this domain. Our experiments with the “infectious” cDNA clone of TMV to assess the role of the carboxy-terminal region of the MP were complicated by the fact that the origin for virus assembly and the subgenomic promoter for the coat protein gene both overlap the coding sequence for the MP gene. We attempted to circumvent this problem by using a TMV vector that contained these sequences independent of the MP gene tested (pU3/12AM-RV). Unfortunately, the presence of repeated sequences between some of the MP deletion mutants and the vector resulted in deletion of these sequences during RNA replication and the reconstruction of the wild-type MP gene. Sequence deletion was first suspected when the MP produced in plants infected with AMCTSwas observed to have the same mobility in SDS-PAGE as wild-type MP. Reverse transcription, PCR cloning, and sequence analysis established that there was reversion to the wild-type sequence. This observation is similar to that reported by Lehto and Dawson (1990). In their experiment a TMV virus carrying a second copy of the MP gene (including a repeat of 8 1 1 nucleotides) reverted to wild-type virus. The deletion process and reconstruction of wild-type sequence of AMCT3(a repeat of 139 nucleotides) apparently occurred more rapidly than the deletion of 81 1 nucleotides observed by Lehto and Dawson (1990). The wild-type MP was detected in inoculated leaves 96 hr after infection by AMCT3 (Fig. 3B), while the virus carrying two copies of the MP gene was stable for more then 10 days (Lehto and Dawson, 1990). The rapid reversion of AMCT3 may reflect the impaired activity of the CT3 mutant, resulting in a wild-type virus population that dominates cell-to-cell movement. The analysis of amino-terminal deletions in the MP gene revealed that a deletion of three amino acids (residues 3-5; the smallest deletion tested) resulted in a nonfunctional protein. It is assumed that such a small
deletion at the amino terminus did not result in a gross structural change, and that the effect is directly on an important determinant for protein stability or activity. The amino terminus of the MP apparently does not function as a cleavable signal peptide (C. M. Deom and R. Gafny, unpublished). The analysis of the movement protein of TMV and other viruses will require genetic, cellular, and biochemical studies. Those described here represent an important first step in a future indepth study of the TMV MP.
ACKNOWLEDGMENTS We are grateful to M. Dyer and S. Leitner for growth and maintenance of plant materials. We also thank colleagues at the Monsanto Company for supplying oligonucleotides for mutagenesis during the early phase of this work. This work was supported by grants from the National Science Foundation to R.N.B. (DMB8717012), the Monsanto Company to R.N.B., and the Center for Plant Science and Biotechnology founded by the Missouri Centers for Advanced Technology. R.G. and M.L. were supported by BARD Fellowships.
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