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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC Jul 12, 2010.
Published in final edited form as:
PMCID: PMC2902183
NIHMSID: NIHMS31774

A conserved binding site within the Tomato Golden Mosaic Virus AL-1629 promoter is necessary for expression of viral genes important for pathogenesis

Abstract

We have identified a nine base pair sequence in Tomato golden mosaic virus that is required for binding of nuclear proteins from tobacco and Arabidopsis to viral DNA. The sequence is located within the promoter for a 0.7 kb complementary sense mRNA (AL-1629). Mutation of the binding site results in a two to six-fold reduction in the accumulation of AL-1629 mRNA, leading to reduced AL2 and AL3 gene expression. Viral sequences located immediately adjacent to the core binding site appear to influence AL2 and AL3 expression, but retain some binding affinity to a soluble host protein(s). The ability of a nuclear protein(s) to bind sequences within the AL-1629 promoter correlates with efficient viral DNA replication, as mutation of these sequences results in reduced viral DNA levels. Analysis of begomo- and curtoviruses indicates extensive conservation of this binding site, which suggests a common mechanism regulating expression of two viral genes involved in replication and suppression of host defense responses.

Keywords: Geminivirus, regulation, promoter, transcription, nuclear extract, binding

Introduction

Geminiviruses from the genus Begomovirus have small genomes, ~2.5 to 3.0 kb in size, and replicate in the nuclei of infected plant cells producing double-stranded (ds) DNA replicative form (RF) intermediates (Preiss and Jeske, 2003; Stenger et al., 1991). Viral RF DNA provides a template for transcription, but these viruses do not encode an RNA polymerase. Therefore, geminiviruses rely heavily on the host transcription machinery for viral gene expression (Bisaro, 1996; Hanley-Bowdoin et al., 2000). This makes geminiviruses a valuable model system for the study of transcription in plants.

The genome of Tomato golden mosaic virus (TGMV) consists of two DNA chromosomes (A and B), both of which are required for infectivity (Hamilton et al., 1983). DNA A (Fig. 1) encodes functions required for replication (AL1 and AL3) and encapsidation (AL2 and AR1) of the virus (Rogers et al., 1986; Sunter et al., 1987). Genes on the B component (BR1 and BL1) encode functions required for viral movement (Brough et al., 1988; Jeffrey et al., 1996). A 5′ intergenic region of ~230 bp (conserved in both components) separates divergent coding regions, and contains elements mediating bi-directional transcription of virus-specific mRNAs (Fontes et al., 1994; Hanley-Bowdoin et al., 1990; Petty et al., 1988; Sunter and Bisaro, 1989; Sunter et al., 1989; Sunter et al., 1993). In contrast to viral sense transcription, the complementary sense transcription unit of TGMV DNA A is complex, consisting of multiple overlapping RNAs with different 5′ ends, all of which are 3′ co-terminal, and potentially multicistronic (Hanley-Bowdoin et al., 1988; Sunter and Bisaro, 1989). The only RNA capable of producing a functional AL1 protein initiates at nucleotide 62 (AL-62), and two smaller RNAs, initiating at nucleotides 1935 and 1629 (AL-1935, AL-1629), specify both the AL2 and AL3 coding regions (Fig. 1). AL-62, AL-1935 and AL-1629 express AL3, but only AL-1629 expresses AL2 (Hanley-Bowdoin et al., 1989; Shung et al., 2006).

Fig. 1
Genome organization of TGMV DNA A. The circular map illustrates the wild type TGMV DNA A genome component, indicating relevant restriction sites with nucleotide coordinates given in parentheses. Numbering is according to the TGMV sequence determined by ...

A minimal sequence has recently been identified that is important for transcription of AL-1629 mRNA, which is located between −129 and −213 nucleotides (nt) upstream of the transcription start site for AL-1629 (Shung et al., 2006). Expression of AL2 and AL3 from the AL-1629 viral transcript requires repression of AL62 transcription, regulated through binding of AL1 protein to sequences in the origin of replication (Shung and Sunter, in press). A region of the AL-1629 promoter sequence between −129 (ApaI) and −184 (ClaI) is capable of binding plant nuclear proteins, although this fragment alone is unable to activate expression from a heterologous promoter (Shung et al., 2006). One possible explanation, which we favor, is that a single sequence element that spans the ClaI restriction site is responsible for expression of AL-1629. An alternative is that two elements contribute to activation, one between −213 to −184 (ClaI) and one between −184 (ClaI) and −129 (ApaI). Similar regions of viral DNA in African Cassava mosaic virus (ACMV) and Mungbean yellow mosaic virus (MYMV) also direct expression of AC2 and AC3 (Shivaprasad et al., 2005; Zhan et al., 1991). Geminivirus RF DNA is associated with host nucleosomes as a viral minichromosome, and one nucleosome-free gap detected in Abutilon mosaic virus (AbMV) co-localized with sequences corresponding to the TGMV AL-1629 promoter (Pilartz and Jeske, 1992; 2003). This would presumably allow interactions between the promoter and additional host factors for transcription of AL-1629.

The role of host factor binding in transcription of geminivirus mRNA in general, and in AL-1629 mRNA expression specifically is currently unknown. In this article, we present results that allow us to identify a core sequence that is required both for binding of host nuclear factors, and is necessary for expression of genes critical for viral pathogenesis.

Results

Identification of a core binding sequence within the TGMV AL-1629 promoter

Previous results had identified a 55 bp sequence, between −129 (ApaI) and −184 (ClaI) upstream of the AL-1629 transcription start site that is capable of binding plant nuclear proteins (Shung et al., 2006). Initially we performed in vitro footprinting experiments using a soluble protein fraction extracted from purified nuclei isolated from Nicotiana benthamiana suspension cells. A region of viral DNA corresponding to sequences surrounding the ClaI site was protected from digestion (data not shown), and supports our previous findings that these sequences are important for AL-1629 promoter activity (Shung et al., 2006).

Based on these results we performed electrophoretic mobility shift assays (EMSA) using a series of three overlapping double-stranded (ds) oligonucleotides that spanned a 60 bp region (nt 1851 to nt 1792) upstream of the transcription start site for AL-1629 and including the ClaI site (Fig. 1). Each dsDNA oligonucleotide comprised 30 bp of TGMV sequence, which overlapped by 15 bp (Table 1). Soluble nuclear proteins bound to dsDNA containing sequences from nt 1792-1821 (oligo A) and from nt 1807-1836 (oligo B), as evidenced by a shift in the labeled probe fragment (Figs. 2A and 2B). Binding of nuclear proteins to both dsDNA fragments is dependent on protein concentration (data not shown). Two products were observed for both oligonucleotides, which could reflect binding of different amounts of a single protein, or binding of multiple proteins. No binding was detected in experiments using dsDNA containing sequences from nt 1851 to 1822 (oligo C; data not shown). Specificity of binding was shown by competition using various dsDNAs as unlabeled competitor. Binding to oligo A was efficiently competed with increasing amounts (100 molar excess) of unlabeled oligo A and oligo B (Fig. 2A, lanes 4-5), but not using unlabeled oligo C (Fig. 2A, lane 3). A similar result was obtained using oligo B, which suggests that the same nuclear protein(s) is responsible for the binding observed upon incubation with both oligo A and B. Binding was not competed using single stranded DNA corresponding to oligo A, B or C (Figs. 2A and 2B, lane 6) demonstrating that the binding detected was not a consequence of any residual single-stranded (ss) DNA left from the purification procedure. Also, binding was not competed with a non-specific 78 bp DNA fragment from pUC118 at a 200 molar excess (Figs. 2A and 2B, lane 7). From this we conclude that TGMV DNA sequences between nt 1836 and 1792 specifically bind a nuclear protein(s), possibly a host transcription factor, consistent with this sequence being the AL-1629 promoter. As oligos A and B are both specific competitors for binding, the binding site most likely resides between nt 1821 and 1807, although it is possible that the binding site extends beyond nt 1821.

Fig. 2
The TGMV AL-1629 promoter region specifically binds nuclear extracts. The linear map represents the region of TGMV viral DNA containing the AL-1629 promoter upstream of the AL2 and AL3 coding regions. Soluble nuclear extracts (S100) isolated from tobacco ...
Table 1
Oligonucleotides used for DNA:Protein binding and protoplast assays

Mutational analysis identifies nucleotides within the AL-1629 promoter required for binding of nuclear proteins

To further analyze sequences within the TGMV AL-1629 promoter that bind plant nuclear proteins, a series of 20 bp dsDNAs was generated spanning sequences between nt 1834 and 1809. Each DNA contained a six bp mutation overlapping by three bp (Table 2), and binding of plant nuclear proteins to these dsDNAs was determined by EMSA. As shown (Table 2), nuclear proteins bound dsDNA containing wild type sequences from nt 1828 to 1809. Mutation of sequences from nt 1827 to 1813 contained within oligos M5, M1, M2 and M3 respectively, abolished binding, whereas mutation of sequences from nt 1815 to 1810 (M4) and from nt 1830 to 1825 (M6) had no apparent effect on binding. As DNAs containing mutations in M3 and M5 overlap with M4 and M6 respectively, which exhibit binding, our conclusion is that a nine bp sequence from nt 1824 to 1816 (AACGTCATC) within the TGMV AL-1629 promoter is required for binding of plant nuclear proteins. However, we cannot rule out that sequences on either side could play a role in stabilizing binding, but this has yet to be tested.

Table 2
Mutational analysis of the DNA binding site within the TGMV AL-1629 promoter

To confirm the specificity of binding we tested the ability of each mutant oligo to compete for binding of nuclear extracts to dsDNA containing wild type sequences (Fig. 3A). As seen, binding to wild type dsDNA was efficiently competed using unlabeled wild type (Fig. 3A, lane 3), or M6 mutant dsDNA (Fig. 3A, lane 9) as competitor. This is consistent with the ability of these DNAs to bind nuclear proteins (Table 2). In contrast, binding was not efficiently competed using M2, M3, or M5 dsDNAs (Fig. 3A, lanes 5, 6 and 8), confirming results that demonstrate these DNAs do not bind nuclear proteins (Table 2). However, partial competition was observed using M1 and M4 dsDNAs (Fig. 3A, lanes 4 and 7), which suggests that these DNAs might exhibit weak binding. Again, no competition was observed using pUC118 (Fig. 3A, lane 10). These experiments were repeated using soluble nuclear extracts isolated from Arabidopsis plants, and similar results were observed (Fig. 3B). Efficient competition for binding of Arabidopsis nuclear proteins was observed upon incubation with wild type, M4 and M6 dsDNAs (Fig. 3B, lanes 4, 8 and 10). This correlates with the ability of these DNAs to bind tobacco nuclear proteins (Table 2). No competition was detectable under these conditions using M1, M2, M3 or M5 as competitor DNAs (Fig. 3B, lanes 5, 6, 7 and 9). The data are again consistent with a nine bp sequence of TGMV DNA (nt 1824 to 1816) within the AL-1629 promoter being essential for binding of nuclear proteins in both tobacco and Arabidopsis. We also see binding to nuclear proteins in extracts isolated from spinach and tomato (data not shown).

Fig. 3
Mutations within the AL-1629 promoter inhibit binding of nuclear extracts. Soluble nuclear extracts (S100) isolated from tobacco suspension cells (A) or Arabidopsis leaves (B), were incubated with 32P-labeled dsDNA containing wild type TGMV DNA sequences ...

The DNA binding site within the TGMV AL-1629 promoter regulates expression of AL-1629 mRNA

To directly assess the importance of binding to sequences within the AL-1629 promoter on transcription, we introduced six bp mutations (M2, M4, M5 and M6) into a promoter-reporter construct, containing 790 bp of TGMV sequence upstream of the AL3 coding region. Previous data has demonstrated that transcription initiating at nt 1935 is extremely low, and that AL-1629 is the predominant RNA expressed in these promoter-reporter constructs (Shung et al., 2006). Promoter-reporter constructs containing each mutation were transfected into protoplasts and total RNA isolated three days post-transfection as described (Shung et al., 2006). Following semi-quantitative RT-PCR, samples were analyzed by DNA gel blot hybridization to a TGMV-specific probe. As a control, RT-PCR was performed using primers designed to amplify a 150 bp fragment of EF1α (Yang et al., 2004), and in all cases the predicted fragment was detected (Fig. 4). Mutations introduced into nt 1827 to 1822 (M5) or nt 1821 to 1816 (M2) resulted in an approximate three to six-fold decrease in the amount of RNA that accumulated. Mutations introduced on either side of this sequence, M6 (nt 1830 to 1825) or M4 (nt 1815 to 1810), led to a two- to three-fold reduction in the accumulation of RNA. These results correlate with the binding assays in that mutations within M2 and M5 abolish binding and lead to a significant decrease in transcription. Mutation within M4 and M6 decrease transcription to a lesser degree which correlates with results demonstrating that binding to these DNAs is also somewhat impaired relative to M2 and M5 (Fig. 3).

Fig. 4
Identification of TGMV RNA in protoplasts transfected with wild type and mutant DNA templates. The panel illustrates phosphorimager analysis of RT-PCR products after 24 cycles of amplification. Aliquots of cDNA were fractionated on agarose gels and hybridized ...

The DNA binding site within the TGMV AL-1629 promoter regulates AL2 and AL3 gene expression

To assess the importance of binding to the sequence located between nt 1824 and 1816 on expression of viral genes, we transfected protoplasts with a series of promoter-reporter constructs. Extracts were prepared three days post-transfection, and fluorometric GUS assays performed as described (Sunter and Bisaro, 1991). Cloned DNA containing 622 bp of TGMV sequence upstream of the transcription initiation site for AL-1629 directs high levels of AL2/GUS (AL2 [−654]-GUS) and AL3/GUS (AL3 [−790]-GUS) expression (Shung et al., 2006). Mutation of sequences between nt 1827 and 1810 (M2, M4, and M5) results in a significant reduction in AL2 and AL3 expression (Student’s t-test: P<0.05) as compared to wild type (Fig. 5A and 5B). The greatest reduction was observed when sequences between nt 1821and 1816 (M2) were mutated which correlates with results demonstrating that this mutation leads to loss of binding of plant nuclear proteins (Table 2) and the greatest decrease in RNA accumulation (Fig. 4). Mutation of sequences from nt 1815 to 1810 (M4) and nt 1827 to 1822 (M5) led to a two- to five-fold decrease in AL2 and AL3 expression (Fig. 5A and 5B), similar to the observable decrease in RNA accumulation (Fig. 4). Mutation of sequences between nt 1830 and 1825 (M6) resulted in no significant decrease in AL2 expression (Fig. 5A), but an approximate two-fold reduction in AL3 expression (Fig. 5B).

Fig. 5
Expression of AL2 and AL3 from promoter-reporter constructs containing mutations within the TGMV AL-1629 promoter region. The ability of TGMV AL-1629 promoter mutations to direct expression of TGMV AL2 and AL3 was determined by comparing GUS activity ...

The templates used in these experiments contain the transcription initiation site for both AL-1629 and AL-1935, although AL-1629 is the predominant RNA expressed in these promoter-reporter constructs, and transcription initiating at nt 1935 is extremely low (Shung et al., 2006). We therefore decided to confirm these results by introducing two of these mutations into a promoter-reporter construct that only contains the AL-1629 transcription initiation site, but still expresses both AL2 and AL3 (Shung et al., 2006). As can be seen (Fig. 5C), mutation of sequences between nt 1821and 1816 (M2) again led to a significant decrease (Student t-test: P<0.05) of five to ten-fold in AL2 and AL3 expression respectively. A smaller, but still significant (Student t-test: P<0.05) decrease in expression was also detected when sequences between nt 1815 to 1810 (M4) were altered (Fig. 5C). Together, the results demonstrate that mutation of sequences required for AL-1629 transcription results in a decrease in AL2 and AL3 expression.

DNA binding sequences within the AL-1629 promoter are important for efficient TGMV DNA replication in protoplasts

To assess the biological importance of the viral sequence shown to be important for AL2 and AL3 gene expression, one of the six bp mutations (M4) was introduced into a TGMV DNA background capable of independent replication (pTGA26; Sunter et al., 1990). We chose M4 on the basis that this mutation results in decreased binding and AL-1629 transcription. The mutation does not disrupt the TGMV AL1 coding region but results in amino acid substitutions from two aspartate residues (amino acids 263 and 264) in the wild type protein to serine and threonine in the predicted M4 mutant protein. The effect of this change, if any, on viral replication is unknown, although the change is in the helicase domain of the AL1 protein (Hanley-Bowdoin et al., 2000). As shown, introduction of the M4 mutation results in an approximate four-fold reduction in replicating viral DNA (Fig. 6, lane 2) as compared to wild type (Fig. 6, lane 1). As this could be due to loss of AL1 function as a result of changes in amino acid sequence, we co-transfected protoplasts with mutant DNA along with DNA capable of expressing AL1 (pTGA73; Sunter et al., 1993). The addition of exogenous AL1 protein did not increase replicating viral DNA levels derived from the M4 mutant DNA template (Fig. 6, lane 3). As previous data has demonstrated reduced replication in the absence of AL3 (Sunter et al., 1990), we tested whether the defective replication phenotype observed in M4 mutant DNA is due to loss of AL3 expression. Protoplasts were co-transfected with mutant DNA and a construct known to complement a deficiency in AL3 (pTGA81; Sunter et al., 1990). Unfortunately, addition of exogenous AL3 alone did not complement the M4 mutation (Fig. 6, lane 4), but an increase in viral DNA levels was observed when exogenous AL1 (pTGA73) and AL3 (pTGA81) were provided together (Fig. 6, lane 5). These results suggest that the mutant phenotype observed upon the introduction of mutation M4 is a consequence, in part, of the lack of AL3 and most likely AL-1629 transcription.

Fig. 6
Mutation of the AL-1629 promoter affects TGMV replication. The panel illustrates phosphorimager analysis of DNA (1 μg) isolated from protoplasts transfected with either wild type, or mutant, TGMV DNA A. Samples were fractionated on agarose gels ...

Discussion

TGMV encodes three overlapping ORFs that are expressed from multiple complementary sense RNAs (Hanley-Bowdoin et al., 1988; Sunter and Bisaro, 1989). A single RNA can produce a functional AL1 protein (AL-62), and a single smaller RNA (AL-1629) can express AL2 (Shung et al., 2006). Using electrophoretic mobility shift assays, we have identified a nine bp core sequence surrounding the ClaI site (Fig. 1), located between nt 1824 to 1816 (AACGTCATC), that specifically binds a nuclear protein(s), which may reflect a host transcription factor(s). The nine bp sequence is located between 187 and 195 bp upstream of the transcription initiation site for AL-1629, and is contained within the sequence shown to be necessary for AL-1629 transcription (Shung et al., 2006). The relevance of this core sequence to viral gene expression was shown by mutational analysis, where sequences between 1824 and 1810 are required for maximal expression of AL2 and AL3, and alteration of this core sequence resulted in up to a five-fold decrease in AL-1629 RNA accumulation. These results correlate with DNA:Protein binding assays in that mutations that lead to a significant decrease in transcription also abolish binding of plant nuclear proteins. Similarly, mutations that lead to a smaller decrease in transcription exhibit impaired binding (Fig. 3). Taken together, our results suggest a core sequence of six bp (nt 1821 to 1816) is essential for AL2 and AL3 expression via AL-1629 transcription, and nucleotides immediately adjacent play an important role in enhancing expression, possibly through stabilizing binding. However, this is difficult to test using nuclear extracts, which may contain multiple DNA binding proteins.

Alteration of the binding sequence from nt 1815 to 1810 (M4) resulted in a mutant virus that was unable to replicate efficiently in a tobacco protoplast system. The phenotype observed was similar to that of an AL3 mutation in which replication of viral DNA is reduced at least 50-fold (Sunter et al., 1990). This is consistent with data that shows reduced AL-1629 transcription, and AL3 expression, from DNA templates containing this mutation. It should be noted that AL2 is also expressed from AL-1629 (Shung et al., 2006), but an AL2 mutation results in reduced ssDNA accumulation and wild type levels of dsDNA (Sunter et al., 1990). Therefore, this phenotype would be masked by the more debilitating AL3 mutation. The observable phenotype was not exclusively due to lack of AL3 expression, as both AL1 and AL3 were required to complement the mutation. Although the M4 mutation affected replication, it had no observable affect on the ability of AL1 to autoregulate its own expression (data not shown). This is important as we have recently demonstrated that AL-1629 transcription, and hence AL2 and AL3 expression, is dependent on AL1 autoregulation (Shung and Sunter, in press). Thus, the phenotype observed with the M4 mutation is not a consequence of reduced AL-1629 transcription due to lack of AL1-dependent repression. The DNA templates used for analyzing the effect of mutations on replication contain both the AL-62 and AL-1935 transcription initiation sites, and although AL3 can be expressed from both of these transcripts (Hanley-Bowdoin et al., 1989; Shung et al., 2006), our data suggests the virus requires expression of AL3 from AL-1629 for efficient replication. Thus, AL-1629 transcription in TGMV is critical for expression of both AL2 and AL3, which play crucial roles in viral pathogenesis (Hao et al., 2003; Sunter et al., 2001; Wang et al., 2003).

A minimal promoter sequence located between −129 and −213 nucleotides upstream of the transcription start site for AL-1629 has recently been identified (Shung et al., 2006), and similar regions of viral DNA in African Cassava mosaic virus (ACMV) and Mungbean yellow mosaic virus (MYMV) also appear to direct expression of AC2 and AC3 (Zhan et al., 1991) (Shivaprasad et al., 2005). A comparison of sequences upstream of the AL2 and AL3 coding regions in a number of begomoviruses illustrates a remarkable degree of identity (Fig. 7). The 12 bp sequence required for efficient expression of TGMV AL2 and AL3 is 100% conserved in all begomoviruses analyzed, with the exception of a single nucleotide within the TGMV sequence. This sequence is located ~288 nt upstream of a transcription initiation site mapped in MYMV (Shivaprasad et al., 2005), that would produce a transcript comparable to TGMV AL-1629. Analysis of sequences upstream of two curtoviruses also indicates that this 12 bp sequence is 100% conserved (Fig. 7). In Spinach curly top virus (SCTV) this sequence is located 30-60 bp upstream of initiation sites for complementary sense transcripts encoding C2 and C3 (Baliji et al., 2007). This sequence is also capable of directing expression of C2 in a promoter-reporter fusion (data not shown). The ability of this element to bind nuclear proteins in extracts isolated from Arabidopsis, spinach, tobacco and tomato is consistent with conservation of this sequence in viruses that infect these hosts.

Fig. 7
Alignment of 5′ flanking sequences of the AL2 and AL3 coding regions from selected begomo- and curtoviruses. The viruses used in the alignment were Tomato golden mosaic virus (TGMV; GenBank accession no. ...

Putative binding sites for ATF1, TGA2 and v-Myb and c-Myb (Fig. 7) were identified using Transcription Element Search Software (TESS: http://www.cbil.upenn.edu/tess). ATF is involved in expression of adenovirus E1a-inducible genes and binds elements in cellular cAMP-inducible promoters (Lin and Green, 1988). TGA2 is a basic leucine zipper protein that interacts with salicylic acid and auxin response elements (Lam and Lam, 1995), and is part of the DNA binding activity of Activating Sequence Factor 1 found in monocot and dicot plants. Myb factors regulate expression of genes involved in apoptosis, cell proliferation, and defense responses (Araki et al., 2004; Chen et al., 2006; Denekamp and Smeekens, 2003). Binding sites for v-Myb and c-Myb might suggest that AL2 and AL3 expression could be dependent on pathways involved in regulation of cell cycle and/or defense, although there is currently no evidence for this. However, conserved binding sites located upstream of the AL2 and AL3 coding regions in begomo- and curtoviruses (Baliji et al., 2007; Shung et al., 2006) suggests a common mechanism regulating expression of two viral genes involved in replication and suppression of host defense responses (Hao et al., 2003; Wang et al., 2003; Wang et al., 2005).

We are currently performing genetic screens to identify the host factor(s) that regulates AL-1629 transcription. Demonstration that the core sequence binds nuclear extracts from Arabidopsis, and conservation amongst geminiviruses that infect Arabidopsis, provides confidence that this approach will be successful. Identification of the host factor(s) regulating transcription of these important genes might allow the development of broad range approaches to control of an important group of plant pathogens through interference of the interaction.

Materials and Methods

DNA techniques

The map locations and restriction endonuclease sites cited here refer to the published DNA sequence of TGMV (Hamilton et al., 1984). All restriction endonucleases and DNA modifying enzymes were used as recommended by the manufacturers. DNA and RNA manipulations, polymerase chain reaction, and electrophoretic mobility shift analyses were performed essentially as described by (Ausubel et al., 2001) unless otherwise stated.

Electrophoretic mobility shift analysis

Pairs of complementary oligonucleotides containing TGMV sequences were synthesized (Invitrogen, Carlsbad, CA), annealed and dsDNA purified by gel isolation (Ausubel et al., 2001). Oligonucleotides contained either wild type TGMV sequences or a series of six bp mutations flanked by wild type sequences. The mutations in each oligonucleotide overlapped by three bp (Table 1). Soluble protein extracts (S100) were isolated from Arabidopsis, N.benthamiana, spinach or tomato nuclei as described (Shung et al., 2006), and binding of nuclear proteins to dsDNA oligonucleotides examined by electrophoretic mobility shift assay (EMSA). Radiolabeled probe DNA was incubated with 10 to 15 μg soluble nuclear protein extracts, 375 ng poly (dI:dC), 50 fmol probe, and 5 pmol competitor DNA in 15 μl binding buffer (50 mM Tris-HCl, pH7.5, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 20% glycerol). Reactions were incubated at room temperature for 30 minutes, and complexes resolved by electrophoresis in a 6% polyacrylamide DNA retardation gel (Invitrogen, Carlsbad, CA). The gel was dried by vacuum onto Whatman 3M paper prior to exposure using a phosphor storage screen and analyzed using a phosphorimager (Biorad, Hercules, CA).

Promoter-reporter constructs

Promoter-reporter constructs containing a translational fusion between the β-glucuronidase reporter gene (GUS) and the N-terminal 83 amino acids of the AL2 coding region, or the N-terminal 36 amino acids of the AL3 coding region have been described. DNAs contained either 622 bp (AL2[-644]-GUS and AL3[-790]-GUS) or 216 bp (AL2[-244]-GUS and AL3[-380]-GUS) of 5′ flanking sequence, upstream of the transcription start site for AL1629 (Shung et al., 2006). A fragment of TGMV DNA A containing 778 bp from the EcoRI site to the AL3 translation start site (Fig. 1) was inserted into the pGEM4 vector (Promega, Madison, WI) to generate a holding plasmid (pGS251). Mutagenic primers (M2 and M4; Table 2) were used to introduce six bp mutations into this DNA by site-directed mutagenesis according to the manufacturers’ instructions (QuikChange II Site-directed Mutagenesis Kit, Stratagene, La Jolla, CA). Mutations M5 and M6 were generated by PCR using pGS251 as template with either primers M5-R and AL2-5A, or M6-R and AL2-5A (Table 1). The resulting PCR products were digested with EagI and ClaI, and the 150 bp fragment used to replace the corresponding wild type fragment of pGS251. The resulting DNA was then restricted with EagI and ApaI, and the resulting 205 bp DNA fragment containing each mutation used to replace the corresponding wild-type fragment of each promoter-reporter construct (Shung et al., 2006). The presence of all mutations was confirmed by sequencing of individual clones (Plant-Microbe Genomics Facility, The Ohio State University).

Cloned DNAs capable of generating a replicating TGMV genome component (pTGA26), and or constitutively expressing AL1 (pTGA73) or AL3 (pTGA81) from the CaMV 35S promoter, have been previously described (Sunter et al., 1990). DNA containing the M4 mutation was restricted with EagI and ApaI, and the resulting 205 bp DNA fragment the mutation used to replace the corresponding wild-type fragment of pTGA26.

Protoplast transfection and analysis

Protoplasts were isolated from an N.benthamiana suspension culture cell line and transfected with various DNAs and fluorometric GUS assays performed after three days as described (Sunter and Bisaro, 2003). GUS activities were compared using Student’s t-test.

RNA isolation and analysis

Total RNA was isolated from N.benthamiana protoplasts using RNeasy Plant Mini Kit according to the manufacturers’ instructions (Qiagen, Valencia, CA) and quantified by spectrophotometer. Up to 500 ng total RNA was used for detection of EF1α or TGMV viral RNAs (Table 1) in semi-quantitative reverse transcription (RT)-PCR reactions using SuperScript one-step RT-PCR mix with Platinum Taq according to the manufacturers instructions (Invitrogen, Carlsbad, CA). Products from RT-PCR reactions were electrophoresed through 1% TAE agarose gels, transferred to Protran® pure nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and immobilized by UV cross-linking (UV-Strata linker 1800, Stratagene, La Jolla, CA). Specific cDNA products were detected by hybridization to 32P-labeled probes specific for either TGMV or EF1α, generated by random priming (DECA Prime II labeling kit. Ambion, Austin, TX). DNA levels were quantified by phosphorimager analysis (Molecular Imager FX, Bio-Rad, Hercules, CA).

DNA isolation and analysis

DNA was isolated from transfected protoplasts as described previously (Baliji, Sunter, and Sunter, 2007) and quantified by spectrophotometer. Total DNA (2 μg) was electrophoresed through 1% agarose gel, transferred to Protran nitrocellulose transfer membrane (Schleicher & Schuell, Sanford, ME), and immobilized by UV cross-linking (UV-Strata linker 1800, Stratagene, La Jolla, CA). TGMV DNA was detected by hybridization with a 32P-labeled probe specific for TGMV DNA A (Sunter et al., 2001).

Acknowledgements

The research was supported in part by a National Institutes of Health MBRS/SCORE Grant (GM-08194). We thank Chia-Yi Shung and Gabriela Lacatus for critical reading of the manuscript, and to Janet Sunter for maintenance and generation of N.benthamiana cell cultures.

Footnotes

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