Skip to main content
NCBI home page
The Plant Cell logoLink to The Plant Cell
. 2003 Dec;15(12):3020–3032. doi: 10.1105/tpc.015180

Adenosine Kinase Is Inactivated by Geminivirus AL2 and L2 Proteins

Hui Wang a, Linhui Hao a, Chia-Yi Shung b, Garry Sunter b, David M Bisaro a,1
PMCID: PMC282852  PMID: 14615595

Abstract

AL2 and L2 are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively. Both are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants. To understand how geminiviruses defeat host mechanisms that limit infectivity, we searched for cellular proteins that interact with AL2 and L2. Here, we present evidence that the viral proteins interact with and inactivate adenosine kinase (ADK), a nucleoside kinase that catalyzes the salvage synthesis of 5′-AMP from adenosine and ATP. We show that the AL2 and L2 proteins inactivate ADK in vitro and after coexpression in Escherichia coli and yeast. We also demonstrate that ADK activity is reduced in transgenic plants expressing the viral proteins and in geminivirus-infected plant tissues. By contrast, ADK activity is increased after inoculation of plants with diverse RNA viruses or a geminivirus lacking a functional L2 gene. Consistent with its ability to interact with multiple cellular kinases, we also demonstrate that AL2 is present in both the nucleus and the cytoplasm of infected plant cells. These data indicate that ADK is targeted by viral pathogens and provide evidence that this “housekeeping” enzyme might be a part of host defense responses. In previous work, we showed that AL2 and L2 also interact with and inactivate SNF1 kinase, a global regulator of metabolism that is activated by 5′-AMP. Together, these observations suggest that metabolic alterations mediated by SNF1 are an important component of innate antiviral defenses and that the inactivation of ADK and SNF1 by the geminivirus proteins represents a dual strategy to counter this defense. AL2 proteins also have been shown to act as suppressors of RNA silencing, an adaptive host defense response. A possible relationship between ADK inactivation and silencing suppression is discussed.

INTRODUCTION

Geminiviruses are single-stranded DNA viruses that infect a wide range of plant species and cause considerable losses of food and fiber products. These relatively simple pathogens amplify their genomes in the nuclei of host cells by a rolling-circle replication mechanism that uses double-stranded DNA intermediates as replication and transcription templates (Bisaro, 1996; Gutierrez, 1999; Hanley-Bowdoin et al., 1999). Because geminiviruses do not encode a DNA or RNA polymerase, viral replication and transcription depend almost entirely on cellular machinery. However, in members of the genus Begomovirus (e.g., Tomato golden mosaic virus [TGMV]), the product of the AL2 gene additionally is required for the expression of late viral genes, including coat protein and the BR1 nuclear shuttle protein that is necessary for the spread of the virus in infected plants (Sunter and Bisaro, 1992). Stimulation of the coat protein promoter in phloem and mesophyll cells involves both activation and derepression by AL2 (Sunter and Bisaro, 1997, 2003). The 15-kD AL2 (also known as transcriptional activator protein) has a C-terminal activation domain that is functional in plant, yeast, and mammalian cells (Hartitz et al., 1999).

Beet curly top virus (BCTV; genus Curtovirus) encodes a positional homolog of AL2 called L2. However, the L2 protein is not required for late viral gene expression and shares only limited identity with AL2 (Stanley et al., 1992; Hormuzdi and Bisaro, 1995). The most conserved region encompasses ∼20 amino acids near the middle of both proteins and contains a series of Cys and His residues that might form a zinc binding structure. Despite their differences, the two proteins share a common function that does not involve transcriptional activation. When expressed from transgenes in Nicotiana benthamiana and Nicotiana tabacum var Samsun plants, both AL21-100 (lacking the activation domain) and L2 condition a unique enhanced-susceptibility phenotype. More specifically, TGMV, BCTV, and the RNA-containing Tobacco mosaic virus exhibit greater infectivity on plants expressing AL21-100 or L2 than they do on nontransgenic plants. Enhanced susceptibility is characterized by a reduction in mean latent period (time to the first appearance of symptoms) and by a decrease in the inoculum concentration required to elicit infection, without significant enhancement of disease symptoms or an increase in virus replication levels (Sunter et al., 2001).

In a recent study, we showed that the enhanced-susceptibility phenotype is attributable to the interaction of AL2 and L2 with SNF1 kinase, a global regulator of metabolism. In response to nutritional or environmental stresses that deplete ATP, SNF1 turns off energy-consuming biosynthetic pathways and turns on alternative ATP-generating systems. AL2 and L2 inactivate SNF1 both in vitro and in vivo. Furthermore, reducing SNF1 activity in transgenic plants by antisense expression causes enhanced susceptibility similar to that conditioned by AL2 and L2 transgenes, whereas SNF1 overexpression leads to enhanced resistance (Hao et al., 2003). Thus, metabolic alterations mediated by SNF1 appear to be a component of innate antiviral defenses, and SNF1 inactivation by AL2 and L2 can be viewed as a counterdefensive measure.

One of the most exciting developments in the field of virus–host interactions in recent years is the recognition that RNA silencing can act as an adaptive response to limit virus replication (Baulcombe, 1999; Carrington et al., 2001; Vance and Vaucheret, 2001; Waterhouse et al., 2001; Ahlquist, 2002). RNA silencing (also known as post-transcriptional gene silencing, RNA interference, and gene quelling) occurs in most eukaryotes and involves the induction, by double-stranded RNA, of a multistep process that leads to RNA-directed, sequence-specific gene silencing. Studies in several model organisms have led to the identification of silencing machinery components and a partial understanding of the silencing process (Bass, 2000; Sharp, 2001; Plasterk, 2002; Zamore, 2002; Tang et al., 2003).

Most plant viruses, including geminiviruses, are both inducers and targets of RNA silencing, and many actively counter this defense by encoding proteins with antisilencing activity (Brigneti et al., 1998; Kasschau and Carrington, 1998; Kjemtrup et al., 1998; Voinnet et al., 1999). Here, the most relevant examples are the AL2 (also called AC2 or C2) proteins from the begomoviruses African cassava mosaic virus (ACMV) and Tomato yellow leaf curl virus (Voinnet et al., 1999; van Wezel et al., 2002). Recent experiments from this laboratory indicate that TGMV AL2 and BCTV L2 also have antisilencing activity (our unpublished results). Thus, AL2 and L2 appear to disable both innate (SNF1-mediated) and adaptive (RNA silencing–mediated) defense pathways.

The mechanisms by which viral proteins suppress RNA silencing are unknown. To investigate this question and how AL2 and L2 counter both adaptive and innate defenses, we searched for interacting cellular proteins. A cDNA corresponding to adenosine kinase (ADK) was selected from a yeast two-hybrid screen of an Arabidopsis cDNA library on the basis of its ability to interact with AL2 and L2. ADK (EC 2.7.1.20; adenosine 5′-phosphotransferase) is an abundant purine nucleoside kinase present in all eukaryotic cells examined to date. In the presence of magnesium, it catalyzes the transfer of γ-phosphate from ATP or GTP to adenosine to produce 5′-AMP. Thus, ADK plays a critical role in the adenine and adenosine salvage pathways and is important for the synthesis of nucleic acids and nucleotide cofactors. By recycling adenosine, ADK also plays a major role in sustaining the methyl cycle and S-adenosylmethionine–dependent methyltransferase activity in both yeast and plants (Lecoq et al., 2001; Weretilnyk et al., 2001; Moffatt et al., 2002). ADK also may regulate the interconversion of cytokinin bases and ribosides (von Schwartzenberg et al., 1998). In addition, 5′-AMP binds and activates plant SNF1 and its mammalian homolog AMP-activated protein kinase (AMPK) (Davies et al., 1995; Hawley et al., 1995; Sugden et al., 1999a).

In this report, we demonstrate that geminivirus AL2 and L2 proteins interact with and inactivate ADK both in vitro and in vivo, that ADK activity is reduced in transgenic plants expressing these proteins, and that ADK activity is reduced in geminivirus-infected plants. By contrast, we show that ADK levels are increased in plants infected with diverse RNA viruses or with a BCTV mutant lacking a functional L2 protein. Finally, we demonstrate that AL2 is present in both the nuclear and cytoplasmic compartments of infected cells. We propose that ADK inactivation helps to suppress SNF1-mediated responses and also may play a role in suppressing RNA silencing.

RESULTS

AL2 and L2 Interact with ADK

Because AL2 and L2 condition enhanced susceptibility when expressed in transgenic plants (Sunter et al., 2001), we were interested in identifying cellular proteins that interact with these related geminivirus proteins. The yeast two-hybrid system used in the search relies on strain Y190, which harbors both HIS3 and lacZ reporter genes (Durfee et al., 1993; Harper et al., 1993). This system was used in an earlier screen to identify the interactions between AL21-83 and L2 with SNF1 (Hao et al., 2003). Continued screening using AL21-115 (lacking the activation domain) and proteins expressed from an Arabidopsis cDNA activation domain fusion library identified a protein corresponding to the C-terminal residues 145 to 345 of ADK, which was designated ADK-C. An Arabidopsis genomic sequence containing ADK-C was mapped to chromosome 5 by Basic Local Alignment Search Tool (BLAST) search. From this, a full-length mRNA sequence was deduced, which in turn was used to design a forward PCR primer that included the predicted start codon and a reverse primer that contained the stop codon. Using an Arabidopsis cDNA library as a template, these primers generated a 1038-bp DNA fragment that contained a full-length ADK sequence (345 amino acids; 37.9 kD) (data not shown). Arabidopsis has two ADK genes, ADK1 and ADK2, which are 92% identical at the amino acid level (Moffatt et al., 2000). The amino acid sequence encoded by the cDNA identified in our screen is identical to that of ADK2.

The interaction of full-length ADK2 with TGMV AL2 and BCTV L2 was confirmed in the two-hybrid system. Yeast growth in selective medium lacking His (indicative of interaction) was supported when cells expressed both full-length ADK2 and either AL21-115 or L2. These cells also were positive in β-galactosidase filter assays, and similar results were obtained regardless of whether the proteins were expressed as bait or prey (data not shown). Interaction also was observed with the AL2 (AC2) protein of ACMV and with AL2 from Cabbage leaf curl virus (CaLCuV), which infects Arabidopsis. However, no growth was evident when ADK2 was coexpressed with noninteracting, negative control proteins, including p53, CDK2, lamin, TGMV AL1, AL3, and coat protein, and BCTV coat protein. We concluded that both AL2 and L2 interact specifically with ADK2.

Not surprisingly, we found subsequently that AL2 and L2 also interact specifically with ADK1 (data not shown). All further work was done with ADK2, which for convenience is referred to simply as ADK.

AL2 and L2 Inhibit ADK Activity in Escherichia coli

E. coli strain HO4 was used in an in vivo assay to verify that the ADK cDNA encoded a functional protein. Prokaryotes do not encode ADK and therefore cannot perform direct phosphorylation of adenosine. Instead, a two-step salvage pathway is used in which adenosine is converted first to adenine by purine nucleoside phosphorylase (deoD) and then ribophosphorylated to AMP by adenine phosphoribosyltransferase. E. coli HO4 (purE, purF, deoD, apt) is deficient in both purine biosynthesis and adenosine salvage (Hove-Jensen and Nygaard, 1989). However, strain HO4 can survive in minimal medium if provided with adenosine and ADK activity. ADK cDNA and truncated ADK cDNAs (N-terminal deletion derivatives ADKΔN13 and ADKΔN88) were inserted into the expression vectors pMAL and pBluescript SK+ (pBSK), and E. coli HO4 cells transformed with these constructs were plated on complete medium and minimal medium containing adenosine. Expression from pMAL produced maltose binding protein fusions, whereas pBSK constructs expressed proteins fused to the β-galactosidase α-peptide. As shown in Figure 1A, none of the constructs had an adverse effect on cell growth in complete medium. However, only cells containing plasmids that expressed full-length ADK (pBSK-ADK and pMAL-ADK) were able to grow in minimal medium containing adenosine. Thus, the ADK cDNA encodes a protein with adenosine kinase activity that is not affected by fusion with the α-peptide or maltose binding protein, and deletion of as few as 13 N-terminal amino acids renders the protein inactive.

Figure 1.

Figure 1.

Open in a new tab

AL2 and L2 Abolish Complementation of E. coli HO4 by ADK.

(A) Complementation of E. coli HO4 growth by ADK. Cells were transformed with the indicated expression plasmids and streaked on complete medium (LB) or minimal medium containing adenosine. Growth was evaluated after incubation at 30°C for 4 days. pMAL and pBSK indicate empty plasmid vectors.

(B) Inhibition of HO4 complementation upon coexpression of ADK with AL2 or L2. Cells were transformed with the indicated expression plasmids. pDHK29 indicates an empty plasmid vector, and pDHK-CAT indicates a plasmid expressing chloramphenicol acetyltransferase (negative control protein).

To determine the consequences of AL2 and L2 interaction with ADK, the kinase and the viral proteins were coexpressed in HO4 cells. To perform these experiments, full-length AL2 and L2, as well as a control protein that does not interact with ADK (chloramphenicol acetyltransferase [CAT]), were expressed from pDHK29 (Phillips et al., 2000). The replication origin of this plasmid is compatible with those of the pMAL and pBSK vectors. As demonstrated in Figure 1B, coexpression of ADK with CAT, AL2, or L2 had no effect on the growth of E. coli HO4 in complete medium. As noted above, the expression of ADK alone (or with CAT) permitted growth on minimal medium containing adenosine. Remarkably, however, coexpression of either AL2 or L2 with ADK prevented growth on the supplemented minimal medium. These results confirm that AL2 and L2 interact with ADK and indicate that these interactions result in the inhibition of ADK activity.

AL2 and L2 Inhibit ADK in Vitro

As an additional confirmation of functional interaction, partially purified proteins were tested in vitro. His-tagged ADK, deletion derivatives (ADKΔN13, ADKΔN24, and ADKΔN88), and CAT were expressed in E. coli BL21 cells and partially purified by nickel–nitrilotriacetic acid agarose chromatography (Figures 2A and 2B). For unknown reasons, cells expressing full-length ADK yielded less total protein, and consequently less His-ADK, than those expressing the deletion derivatives. AL2 and L2 proteins fused to glutathione-S-transferase (GST-AL2 and GST-L2) likewise were expressed in BL21 cells and partially purified by glutathione-agarose chromatography (Hartitz et al., 1999). His-tagged AL2 (His-AL2) was expressed in insect (Sf9) cells from a baculovirus vector (data not shown).

Figure 2.

Figure 2.

Open in a new tab

ADK Expression and in Vitro Activity.

His-tagged ADK, the indicated ADK deletion derivatives, and CAT were expressed in E. coli BL21 cells and partially purified using nickel–nitrilotriacetic acid agarose resin.

(A) Coomassie blue–stained polyacrylamide gel showing the partially purified proteins. ADK protein concentrations were estimated by comparing band intensities with BSA standards.

(B) Immunoblot of the same gel probed with anti-His tag antibody (anti-his tag).

(C) Demonstration of in vitro ADK activity. Reactions contained the substrates adenosine and ATP with 10 to 70 ng of ADK. Products were resolved by thin layer chromatography. An autoradiograph is shown. Control ATP, ADP, AMP, and adenosine (spotted individually at right) were visualized with UV light.

ADK and deletion derivatives were added to reaction mixtures containing the substrates adenosine and γ-32P-ATP, and the conversion of adenosine to AMP was monitored by thin layer chromatography. No activity was detected with the truncated ADK proteins or with CAT (negative control) in this assay (data not shown). However, AMP was generated in reactions containing full-length ADK, and the amount of product was proportional to the amount of protein added over a range of 1 to 70 ng (Figure 2C). The addition of EDTA to 10 mM completely inhibited the activity, confirming the magnesium dependence of the reaction (data not shown).

In reaction mixtures containing 10 ng of ADK, preincubation with AL2 or L2 at a 3:1 molar ratio before the addition of ATP resulted in a substantial reduction in ADK activity (Figure 3A). By contrast, preincubation with the control protein CAT or GST at a 7:1 molar ratio did not reduce the amount of AMP generated. In experiments using 10 ng of ADK and varying amounts of AL2, ADK activity was reduced nearly 90% at a 1:1 molar ratio of AL2:ADK (Figures 3B and 3C).

Figure 3.

Figure 3.

Open in a new tab

Recombinant AL2 and L2 Proteins Inhibit ADK Activity in Vitro.

(A) Autoradiograph of a chromatogram showing AMP generated in ADK reactions preincubated with the indicated proteins before ATP addition, as described in the text.

(B) ADK activity is reduced proportionally by preincubation with increasing amounts of AL2. Reactions contained 10 ng of ADK and varying amounts of His-AL2.

(C) Stoichiometry of inhibition. The graph shows relative ADK activity plotted against increasing His-AL2:ADK ratio. ADK activity (AMP/AMP+ATP) in each reaction was calculated after phosphorimager quantitation of radioactivity in individual spots.

These results demonstrate conclusively that AL2 and L2 interact directly with ADK and inhibit its activity. The stoichiometry of inhibition also is consistent with a mechanism involving direct interaction between the viral proteins and the cellular kinase.

AL2 and L2 Inhibit ADK Activity in Yeast

L2 was coexpressed with ADK in yeast to determine whether the proteins might interact functionally in the context of a eukaryotic cell. The BCTV protein was used in preference to AL2 in this study because expression of the latter can have adverse effects on yeast cell growth (Hartitz et al., 1999).

We first asked whether Arabidopsis ADK can complement a yeast ADK deletion strain (ado1) that displays a slow-growth phenotype (Lecoq et al., 2001). As shown in Figure 4A, growth of the ado1 mutant was much reduced compared with that of the ADO1 parent strain, but growth was not reduced further by the expression of L2. However, the extent and rate of growth were complemented reproducibly to levels slightly greater than those of the parent strain by the introduction of a plasmid expressing Arabidopsis ADK. Thus, the conservation of yeast and plant ADK proteins apparent at the amino acid level (39% identity) reflects a functional conservation that allows the Arabidopsis protein to complement the growth defect of the yeast ADK deletion strain. However, when the deletion strain was cotransfected with expression plasmids containing ADK and L2, a significant reduction in growth, relative to that observed with the expression of ADK alone, was observed. Using the in vitro ADK assay, comparison of crude extracts obtained from the complemented ado1 mutant and from ado1 cells expressing both ADK and L2 confirmed that the presence of L2 resulted in a significant reduction in ADK activity (Figures 4B and 4C). Cells of the complemented mutant strain (ado1 + ADK) contained nearly twice as much ADK activity as cells also expressing the L2 (ado1 + ADK + L2).

Figure 4.

Figure 4.

Open in a new tab

L2 Reduces the Complementation of a Yeast ADK Deletion Strain by Arabidopsis ADK2.

Results from a representative experiment (one of three replicates) are shown.

(A) Complementation of a yeast ADK deletion strain (ado1) by ADK2, and inhibition by L2. Growth of the ado1 mutant strain and the parent strain (BY4741: ADO1, ura, leu) expressing the indicated proteins was assessed in synthetic complete medium lacking uracil and Leu. In all cases, cells were cotransfected with expression plasmids providing Leu prototrophy (YEpL-ADK2 or YEpL) and uracil prototrophy (YEpU-L2 or YEpU). The YEpL and YEpU plasmids express the LacZ α-peptide.

(B) ADK activity is reduced by L2. Crude extracts (300 ng of protein) were obtained from the indicated yeast cells and examined using the in vitro ADK assay described previously.

(C) Relative ADK activity. The graph shows relative ADK activity (from [B]) in extracts from ado1 cells expressing ADK2 alone or ADK2 + L2, relative to BY4741 cells (ADO1). Labeled AMP was quantitated using a phosphorimager.

We concluded from these experiments that Arabidopsis ADK is capable of complementing the growth defect of the yeast ado1 deletion strain and that the complementation is reduced by L2, with a corresponding reduction in ADK activity. These studies provide strong evidence for a functional, in vivo interaction between L2 and ADK.

AL2 and L2 Inhibit ADK Activity in Plants

Two approaches were used to investigate whether AL2 and L2 might inhibit ADK activity in plant cells. One approach took advantage of previously constructed transgenic N. benthamiana lines expressing AL21-100 or L2 from the constitutive 35S promoter (Sunter et al., 2001). Comparable stem pieces (∼3 mm) were obtained from just below the shoot apex from transgenic plants (AL2 line 472-1 and L2 line CTL2-6) and comparable nontransgenic plants at ∼4 weeks after germination. Stem pieces were used because stem extracts contain considerably more ADK activity than comparable amounts of leaf extract. Each sample consisted of pooled tissue from three randomly selected plants, and three to five samples were analyzed for each treatment in each of six independent experiments.

It was first determined that crude stem extracts from nontransgenic plants displayed ADK activity in the in vitro assay and that the level of activity was proportional to the amount of extract added over a range of 100 to 500 ng (data not shown). Within this range, extracts from transgenic plants expressing AL2 or L2 consistently showed a 15 to 30% reduction in ADK activity compared with extracts from nontransgenic plants (Figure 5A). These reductions were significant (P < 0.05) as determined by Student's t test. Furthermore, they did not appear to be caused by reductions in SNF1 activity (which also was reduced by AL2 and L2), because ADK activity was normal in antisense SNF1 plants (line AS-12; data not shown). Because the nontransgenic and transgenic N. benthamiana plants are isogenic except for the presence of an AL2 or L2 transgene, we concluded that AL2 and L2 interaction with ADK, with consequent inhibition of kinase activity, can occur in planta.

Figure 5.

Figure 5.

Open in a new tab

ADK Levels Are Reduced in Transgenic Plants Expressing AL2 and L2 and Also during Geminivirus Infection.

(A) ADK activity is reduced in transgenic N. benthamiana plants expressing AL21-100 and L2. Crude extracts obtained from comparable stem samples from just below the shoot apex were analyzed for ADK activity as described in the text using equal amounts of protein extract from each sample (150 to 400 ng in different experiments). Labeled AMP produced was quantitated using a phosphorimager. The observed differences between nontransgenic and transgenic plants were statistically significant, as determined by Student's t test (P < 0.05). Graph values represent means ± se.

(B) ADK activity is reduced in TGMV- and BCTV-infected plants. Comparable stem samples from just below the shoot apex were obtained from mock-inoculated N. benthamiana plants and plants infected with wild-type BCTV, a BCTV L2 mutant, TGMV, PVX, or CMV and analyzed as described above and in the text. In all cases, the observed differences between virus-infected and control plants proved statistically significant, as determined by Student's t test (P < 0.05). Graph values represent means ± se.

In the second approach, the in vitro ADK assay was used to determine whether kinase activity in stem extracts obtained from TGMV- and BCTV-infected N. benthamiana plants was reduced relative to that in extracts from mock-inoculated plants and plants infected with BCTV containing a frameshift mutation that inactivates L2. The BCTV L2-2 mutant was chosen for this study because it replicates to nearly wild-type levels and generates symptoms similar to those of wild-type virus (Hormuzdi and Bisaro, 1995). TGMV AL2 mutants could not be used in this experiment because they do not express the BR1 movement protein and thus are not infectious (Sunter and Bisaro, 1992). ADK activity also was measured in plants infected with the unrelated, RNA-containing Cucumber mosaic virus (CMV) and Potato virus X (PVX). Stem pieces, which showed no obvious necrosis, were obtained from comparable locations near the shoot apex of infected and mock-inoculated plants. Stem extracts are appropriate for this study because all of the viruses tested here infect both phloem and mesophyll cells except for BCTV, which is phloem limited. Two to three experiments were performed with each virus, and each experiment included three to four replicate samples that consisted of pieces from three randomly chosen plants. Plants were infected at 4 weeks after germination, and tissue was harvested at 14 days after inoculation.

Consistent with previous results, extracts from BCTV- and TGMV-infected plants contained 1.5- and 3.5-fold less ADK activity than extracts from mock-inoculated plants, respectively (Figure 5B). By contrast, ADK activity levels in plants infected with the BCTV L2 mutant virus were 1.3-fold higher than those in mock-inoculated plants. This outcome indicates that functional interaction resulting in the suppression of ADK activity occurs during geminivirus infection and suggests that if the responsible viral protein (L2 in this experiment) is not present, geminivirus infection would cause an increase in ADK activity. The fact that ADK activity also was increased 1.4- to 3.5-fold in PVX and CMV-infected plants supports the idea that virus infection can induce an increase in ADK activity.

AL2 Accumulates in the Cytoplasm and the Nucleus

AL2 is a phosphoprotein that binds single-stranded DNA and zinc and has an acidic, C-terminal activation domain (Hartitz et al., 1999). In keeping with its role in transcription activation, the AL2–green fluorescent protein (GFP) fusion protein has been shown to localize to the nucleus (van Wezel et al., 2001). However, because AL2 appears to interact with and inhibit cytoplasmic ADK in vivo, it was important to demonstrate that some AL2 protein can be found in the cytoplasm.

Several different approaches were used to localize TGMV AL2. First, we observed considerable GFP signal in both the nucleus and the cytoplasm of N. benthamiana and N. tabacum BY-2 protoplasts after transfection with expression plasmids producing AL2 fused to multiple GFP moieties at either the N or the C terminus (AL2-GFP-GFP and GFP-GFP-AL2; data not shown). However, given the considerable cytoplasmic and nuclear signal seen in control transfections with GFP-GFP and GFP-GFP-GFP fusion proteins, we concluded that it was difficult to localize AL2 with this method.

Using AL2 antiserum and a fluorescently labeled (fluorescein isothiocyanate [FITC]) secondary antiserum, we observed that cells from transgenic N. benthamiana plants expressing truncated TGMV AL21-100 displayed widespread fluorescence, which suggested that the protein is present throughout the cell (data not shown). However, because the localization of native protein might be different and additionally might be influenced by other viral proteins or the infection process, we attempted to identify AL2 in TGMV-infected cells. Epidermal peels taken from infected N. benthamiana plants or mock-inoculated plants were examined under comparable conditions using the TGMV AL2 antiserum. As shown in Figure 6, AL2 was found to accumulate in the nucleus of infected cells, as verified by 4′,6-diamidino-2-phenylindole (DAPI) staining. However, in nearly all infected cells, a significant amount of fluorescence, indicating the presence of AL2, also was observed in the cytoplasm, where it appeared in small to large aggregates. At this time, we do not know if these aggregates are associated with specific cellular or virus-induced structures. By contrast, little or no fluorescence was observed in cells from mock-inoculated plants (Figure 6), and no fluorescence was observed in mock-inoculated or infected cells when the primary AL2 antibody was omitted (data not shown). We concluded that AL2 is present in both the nuclear and cytoplasmic compartments in infected cells.

Figure 6.

Figure 6.

Open in a new tab

AL2 Is in the Nucleus and the Cytoplasm of TGMV-Infected Cells.

Epidermal cells from TGMV-infected plants (top three rows) and mock-inoculated plants (bottom row) were examined under bright-field illumination using differential interference contrast optics. Nuclei were located by DAPI staining (blue; left column), and AL2 protein was detected using AL2 primary antiserum followed by FITC-conjugated secondary antiserum (green; middle column). DAPI + FITC merge images are shown in the right column. The irregular cells shown in the top row were originally positioned over mesophyll, whereas the elongated cells shown in the lower rows were associated with veins.

Cell fractionation was another approach used to localize AL2. Native AL2 protein was expressed in insect (Sf9) cells from a baculovirus vector as described previously (Hartitz et al., 1999), and nuclear and soluble cytoplasmic (S-100) fractions were obtained by differential centrifugation. Extracts were prepared 48 h after cells were infected with the baculovirus vector, at which time nuclei remained intact. As shown in Figure 7, AL2 accumulated to significant amounts in both the cytoplasmic and nuclear fractions, and a comparison of relative band intensities suggested that as much as one-third of the AL2 protein was cytoplasmic. It is interesting that the nuclear fraction contained at least three forms of AL2. The two slower migrating species observed in SDS-PAGE were assumed previously to correspond to phosphorylated forms of the protein (Hartitz et al., 1999). Only trace amounts of these forms were present in the cytoplasm, and treatment of nuclear fractions with phosphatase resulted in their disappearance and a concomitant increase in the intensity of the fastest migrating form, which comigrates with cytoplasmic AL2 (Figure 7). Phosphatase treatment of cytoplasmic extracts had no effect on the migration of AL2, further suggesting that this faster migrating form is not phosphorylated (data not shown).

Figure 7.

Figure 7.

Open in a new tab

Localization of AL2 in Insect Cells.

Fractionated protein extracts from Sf9 cells infected with recombinant baculoviruses expressing either native TGMV AL2 or CAT (negative control protein). Cytoplasmic (S-100) and nuclear fractions were prepared at 48 h after inoculation, and equivalent amounts (representing 5 × 105 cells) were electrophoresed on 4 to 20% polyacrylamide-SDS gels and subjected to protein gel blot analysis using an AL2-specific antibody (anti-AL2). AL2 samples also were incubated at 37°C for 3 h with and without calf intestinal alkaline phosphatase (CIAP) before electrophoresis. The positions of nonphosphorylated and phosphorylated AL2 are indicated at right. The two phosphorylated species are indicated by the asterisk.

Given the differential accumulation of phosphorylated and nonphosphorylated forms in the nucleus and the cytoplasm, it is unlikely that the presence of AL2 in the cytoplasm is an artifact caused by the loss of nuclear envelope integrity. And because similar phosphorylated AL2 forms can be observed in whole Sf9 cell extracts (Hartitz et al., 1999), it is unlikely that their relative absence in the S-100 fraction is the result of a compartmentalized phosphatase activity. As a further test, we found no evidence for phosphatase activity directed against AL2 after remixing the S-100 and nuclear fractions (data not shown). Thus, as in infected plant cells, it was concluded that AL2 protein is present in the nucleus and the cytoplasm. In insect cells, phosphorylated AL2 appears to accumulate almost exclusively in the nucleus, whereas nonphosphorylated AL2 is found in both compartments. Because phosphorylation and localization events often occur with high fidelity in insect cells, it is likely that this also is the case in plant cells, although this point remains to be investigated.

DISCUSSION

We showed previously that the TGMV AL2 and BCTV L2 proteins are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants (Sunter et al., 2001). Here, we demonstrate that these two proteins interact with and inactivate ADK in vitro and in vivo and that ADK activity is reduced in geminivirus-infected tissue in a manner that depends, in the case of BCTV, on the presence of a functional L2 protein. We also found that ADK activity is increased in response to infection with CMV, PVX, and a BCTV L2 mutant, suggesting that increasing the activity of this enzyme is part of the host response to infection by RNA and DNA viruses.

The results presented here show that AL2 and L2 interact with ADK. In a previous study, we demonstrated that these same viral proteins also interact with and inactivate SNF1 (Hao et al., 2003). Both ADK and SNF1 are kinases. However, it is unlikely that the AL2/L2 interactions with ADK and SNF1 are mediated solely by a motif conserved among different kinases, because the viral proteins do not interact with cyclin-dependent kinase2 (CDK2) in the yeast two-hybrid system (Hao et al., 2003). In addition, under conditions similar to those used to demonstrate the inhibition of SNF1, AL2 and L2 did not inhibit the in vitro autophosphorylation activity of casein kinase II (data not shown). Finally, AL2 clearly is a substrate for phosphorylation by an as yet unidentified cellular kinase(s) (Hartitz et al., 1999) (Figure 7). Thus, we conclude that the interactions with ADK and SNF1 are both specific and significant and that kinase inactivation is not the only possible consequence of interaction with AL2 and L2.

Our observation, by immunofluorescence microscopy, that AL2 is located in both the nucleus and the cytoplasm of infected N. benthamiana cells provides direct evidence that AL2 has the opportunity to interact with ADK, which is believed to be located primarily in the cytoplasm (Figure 6). At this time, it is not clear whether the localization of AL2 is influenced by other viral proteins, but we did not observe a direct interaction between AL2 and any other TGMV protein in the yeast two-hybrid system (data not shown). The results of cell fractionation studies using extracts from Sf9 cells infected with a baculovirus expressing native AL2 support the conclusion that this protein is present in the nucleus and the cytoplasm and further suggest that phosphorylated forms accumulate predominantly in the nucleus (Figure 7). If confirmed in plant cells, this observation would imply that the functional localization and multiple activities of AL2 are regulated at least in part by cellular protein kinases.

How might a reduction in ADK activity be related to enhanced susceptibility? There are several possibilities, two of which will be discussed here. In eukaryotes, ADK is responsible for recycling adenosine and maintaining intracellular AMP levels. This can be related to our recent finding that AL2 and L2 also interact with and inactivate SNF1 kinase and that the reduction of SNF1 activity in transgenic plants, by expression of an antisense SNF1 transgene, results in enhanced susceptibility (Hao et al., 2003). SNF1 (in plants and yeast) and its mammalian homolog, AMPK, play a central role in the regulation of metabolism in response to stresses that deplete ATP (the cellular stress response) (Halford and Hardie, 1998; Hardie et al., 1998; Johnston, 1999; Hardie and Hawley, 2001). These Ser/Thr kinases exist in heterotrimeric complexes along with two other subunits that serve scaffolding and regulatory functions. When AMP:ATP ratios are increased, SNF1/AMPK complexes turn off energy-consuming systems and turn on alternative ATP-generating systems. In plants, for example, SNF1 has been shown in vitro to phosphorylate and inactivate sucrose phosphate synthase (sucrose synthesis), nitrate reductase (nitrogen for nucleic acid and protein synthesis), and 3-hydroxy-3-methyl glutaryl CoA reductase (steroid and isoprenoid synthesis) (Sugden et al., 1999b).

The available evidence indicates that 5′-AMP binds and activates the plant and mammalian kinases by multiple mechanisms (Davies et al., 1995; Hawley et al., 1995; Sugden et al., 1999a; Hardie and Hawley, 2001). That the AL2 and L2 proteins interact with and inactivate both SNF1 and ADK leads us to advance the proposal that SNF1-mediated metabolic responses are an important component of innate antiviral defense and that geminiviruses have evolved a dual approach to disabling these responses (i.e., by inactivating both SNF1 and ADK) (Hao et al., 2003). Inherent in this model is the idea that AMP generated by ADK is an early activator of SNF1, although it is recognized that SNF1 also responds to AMP that accumulates as a result of ATP depletion. However, ATP depletion caused by the stress of virus replication might occur too slowly to generate a useful AMP signal. Our recent observation that ADK and SNF1 interact in the yeast two-hybrid system provides some support for this view (our unpublished results), and we speculate that ADK and SNF1 might exist in a complex for the purpose of generating a rapid metabolic response. This model also predicts that ADK activity should increase after infection with a virus that does not encode a protein capable of interacting with ADK, as is the case with the BCTV L2 mutant and as may be the case for PVX and CMV (Figure 5). The apparent absence of an ADK-inactivating protein could mean that these RNA viruses have not evolved a mechanism to disable this pathway or that they target a downstream step.

Precisely how SNF1-mediated responses act to limit viral infectivity is not clear, but the ability of an organism to mount an effective defense against an invading pathogen likely depends on the ability of its cells to maintain a positive energy balance. SNF1 shutting off of biosynthetic pathways also might deprive the virus of essential precursors needed for replication. Whatever the mechanism, the fact that geminiviruses target and inactivate both SNF1 and its potential activator ADK underscores the importance of metabolic responses and provides a molecular link between host metabolic status and susceptibility to pathogens. Given the conservation of ADK and SNF1/AMPK, the possibility that metabolic responses are an important feature of pathogen defense in other eukaryotes warrants further investigation.

Another possible connection between ADK and viral pathogenicity is the observation that AL2 (also known as AC2 or C2) from ACMV and Tomato yellow leaf curl virus can suppress RNA silencing (post-transcriptional gene silencing) (Voinnet et al., 1999; van Wezel et al., 2002). We have found that TGMV AL2 and BCTV L2 also are silencing suppressors (our unpublished results). In plants, viruses are both initiators and targets of RNA silencing, which acts to limit the extent of virus infection (Baulcombe, 1999; Carrington et al., 2001; Vance and Vaucheret, 2001; Waterhouse et al., 2001; Ahlquist, 2002). It has been noted that the maintenance of both RNA silencing and transcriptional gene silencing is associated with the methylation of target gene sequences (Bender, 2001; Jones et al., 2001; Paszkowski and Whitham, 2001). RNA silencing also has been associated with specific histone methylation, particularly histone H3 at Lys-9, and histone and DNA methylation may be coupled (Jackson et al., 2002; Volpe et al., 2002). In the case of the geminiviruses, which produce mRNAs from a double-stranded DNA template that assembles into a minichromosome (Pilartz and Jeske, 1992), methylation of the viral genome and chromatin components must be considered as a potential mechanism to limit viral gene expression.

ADK activity is critical to sustain the methyl cycle and S-adenosylmethionine–dependent transmethylation. In yeast, the available evidence suggests that a primary role of ADK is to recycle adenosine produced by the methyl cycle (Lecoq et al., 2001). This also may be true in plant cells, in which ADK activity has been observed to increase (∼1.5- to 3-fold) in response to methyl demand (Weretilnyk et al., 2001). The fact that transgenic, ADK-deficient Arabidopsis plants have reduced transmethylation activity supports this view (Moffatt et al., 2002). Thus, the increase in ADK activity we observed in infected N. benthamiana plants also might reflect an increased demand for transmethylation activity in response to virus challenge (Figure 5). We recognize that S-adenosylmethionine is an important cofactor that could affect many aspects of cellular metabolism relevant to virus replication. However, considering the role of methylation in reinforcing silencing pathways, we speculate that by inhibiting ADK, AL2 and L2 might indirectly suppress silencing by interfering with methylation. In support of this idea, transmethylation deficiency has been cited as a possible explanation for the frequent reversion of ADK-silenced plants (see below) (Moffatt et al., 2002).

Unfortunately, our attempts to produce transgenic plants that would permit a direct examination of the relevance of ADK to geminivirus infection were unsuccessful. After examining a combined total of 49 transgenic N. benthamiana and Arabidopsis plants containing constructs designed to constitutively overexpress the kinase (ADK sense) or underexpress the kinase (ADK antisense and ADK RNAi [RNA interference]), we obtained only one line with altered ADK activity, as determined by the in vitro assay (data not shown). This exceptional Arabidopsis line (At-RNAi-4) produces two types of progeny: one is extremely stunted and has ∼25% of wild-type ADK activity; the second type is less stunted and has ∼60% of wild-type activity. We cannot explain why only one of the transgenic events caused a difference in ADK activity, despite high levels of transgene expression in the case of the sense and antisense lines. But in several respects, our experience has been similar to that of Moffatt et al. (2002), who previously constructed transgenic Arabidopsis ADK lines. These investigators found that all lines examined (eight containing sense and four containing antisense ADK constructs) had reduced ADK activity, suggesting varying degrees of silencing, and that reversion to the wild type was frequent (10 to 15% of plants in each generation). That is, they did not obtain plants with increased activity, and lines with reduced activity were unstable. In addition, plants with <50% of wild-type ADK activity had a characteristic dwarf phenotype, the severity of which correlated with the amount of residual activity. Based on these results, we speculate that ADK activity is tightly regulated and that attempts to artificially alter activity are overcome by post-translational mechanisms, by epigenetic events, or by some combination of these factors. This may explain in part our observation that transgenic AL2 and L2 plants display only modest reductions (15 to 30%) in ADK activity (Figure 5).

Although we were unable to directly examine the relevance of ADK activity to virus infection using transgenic approaches, there are several reasons why we are confident that the inhibition of ADK activity by AL2 and L2 occurs and is an aspect of geminivirus pathogenesis. First, we have demonstrated that AL2 and L2 interact with and inhibit ADK in vitro. Using intact E. coli and yeast cells as well as extracts from geminivirus-infected plants and transgenic plants expressing AL2 and L2, we also have shown that functional interaction occurs in vivo. Second, the interaction is not virus specific. ADK interacts with AL2 from TGMV and CaLCuV (both New World begomoviruses), with AL2 from ACMV (an Old World begomovirus), and with the more distantly related L2 from BCTV (a curtovirus). The fact that the interaction is conserved across the AL2/L2 protein family suggests that it is not fortuitous. Third, ADK presents an interesting target as it produces 5′-AMP, which can activate SNF1. That AL2 and L2 inactivate both SNF1 and ADK seems more than coincidental. In addition, ADK's role in sustaining the methyl cycle suggests another possible reason to target this nucleoside kinase. Finally, we observed that ADK activity is increased after infection of plants with an L2-deficient geminivirus and with RNA viruses belonging to two different families. This finding provides strong evidence that increasing ADK activity is a component of the host response to virus challenge; thus, inhibition of ADK activity by the geminivirus AL2 and L2 proteins most likely is a deliberate counter response.

In conclusion, ADK inhibition by geminivirus AL2 and L2 proteins may be a mechanism to suppress the induction of SNF1-mediated responses, to suppress RNA silencing or other cellular defense pathways that depend on methylation, or both. It will be interesting to learn more about the roles of ADK and SNF1 in host defense and about the roles of AL2 and L2 in viral pathogenesis.

METHODS

Two-Hybrid Analysis

The yeast two-hybrid system was used to identify interactions between AL21-115 and proteins expressed from an Arabidopsis thaliana cDNA library (Durfee et al., 1993; Harper et al., 1993). The cDNA library was obtained from the ABRC (Ohio State University, Columbus). TGMV AL2 was obtained by PCR (forward primer, 5′-GCGGGCGCCATGCGAAATTCGTCTTCC-3′; reverse primer, 5′-GCGGAGCTCCTAAAGTTGAGAAATGCC-3′) and cloned into the NcoI site of pAS2 to produce pAS-AL21-115. NcoI-cleaved pAS2 was previously rendered blunt ended by treatment with the Klenow fragment. BCTV-Logan L2 was obtained by PCR (forward primer, 5′-GCGCCATGGAAAACCACGTG-3′; reverse primer, 5′-GCGGATCCTTATCCAAGTATATCTC-3′) and inserted as an NcoI-BamHI fragment into pAS2 and pACT2 to generate pAS-L2 and pACT-L2. ADK cDNA larger than the original 605-bp partial fragment was obtained by PCR using the Arabidopsis cDNA library as a template with the reverse primer 5′-CAGCTCGAGAAGCTTAGTTAAAGTCGGGCTTCTCAGGC-3′ and the forward primer 5′-GGGTACCTCTAGAATGGCTTCTTCTTCTAA-3′ (full-length ADK) or 5′-GGGTACCTCTAGAATGGGTAACCCACTCCTC-3′ (ADKΔN13). PCR products digested with XbaI and XhoI were inserted into pFastBacHTb (Life Technologies, Rockville, MD) to generate pHT-ADK and pHT-ADKΔN13. The ADK insert was excised from pHT-ADK as an NcoI-XhoI fragment and inserted into pAS2 or pACT to generate pAS-ADK and pACT-ADK. Positive interaction was indicated by the ability of Y190 cells cotransformed with bait and prey constructs (expressed in pAS2 and pACT2, respectively) to grow on medium lacking His and containing 50 mM 3-aminotriazole. The medium also lacked Leu and Trp to ensure the maintenance of expression plasmids. Additional confirmation of interactions was obtained by assessing β-galactosidase activity using a filter-lift assay.

Escherichia coli Complementation Experiments

ADK, AL2, and L2 were expressed in the purine-deficient Escherichia coli strain HO4 (purE, purF, deoD, apt) (Hove-Jensen and Nygaard, 1989), and complementation was assessed by the ability of cells to grow on selective minimal medium (M9 containing 10 μg/mL Met, 1 μg/mL thiamine, 0.2% mannitol, 1 mM isopropylthio-β-galactoside, 100 μM adenosine, and 0.1 μM 2-deoxycoformycin, which prevents deamination of adenosine to inosine). The medium also contained antibiotics appropriate for the maintenance of expression plasmids: ampicillin (100 μg/mL) for pBluescript SK+ (pBSK; Stratagene) and pMAL, and kanamycin (50 μg/mL) for pDHK29. ADK sequence was obtained from pHT-ADK as a SpeI-XhoI fragment and inserted into pBSK to create pBSK-ADK. Inserts from pHT-ADK and pHT-ADKΔN13 were obtained by digestion with XbaI and HindIII and cloned into pMAL-c2x (New England Biolabs, Beverly, MA) to generate pMAL-ADK and pMAL-ADKΔN13. A BamHI-HindIII fragment obtained from pHT-ADK was inserted into pMAL to create pMAL-ADKΔN88. Full-length TGMV AL2 was obtained by PCR (upstream primer, 5′-CGCAGATCTGAATTCATGCGAAATTCGTCTTCCTCA-3′; downstream primer, 5′-GCGGAGCTCCTATTTAAATAAGTTCTC-3′) and inserted as a BamHI-EcoIRI fragment into the BamHI-EcoRV sites of pDHK29 (Phillips et al., 2000) to create pDHK-AL2. BCTV L2 was removed from pAS-L2 as a NcoI-BamHI fragment and inserted into pDHK29 to generate pDHK-L2.

Yeast Complementation Experiments

The Saccharomyces cerevisiae ado1 deletion strain (record number 2583) and the BY4741 parent strain were obtained from Research Genetics (Huntsville, AL) (BY4741: ADO1, MATa, his3D1, leu2D0, met15D0, ura3D0). The ADK sequence was excised from pHT-ADK as a Stu1-XhoI fragment and ligated with SmaI-SalI–cleaved YEpL to create YEpL-ADK. YEpU-L2 has been described previously (Hao et al., 2003). Growth experiments were performed by inoculating synthetic defined medium lacking uracil and Leu with equal numbers of cells from overnight cultures. Cells were incubated at 30°C. In all cases, cells contained expression plasmids providing Leu prototrophy (YEpL-ADK2 or YEpL) and uracil prototrophy (YEpU-L2 or YEpU). The YEpL and YEpU plasmids express the LacZ α-peptide, whose coding sequence was replaced by ADK and L2, respectively.

Protein Expression

His-tagged ADK, various ADK deletion derivatives, and chloramphenicol acetyltransferase were expressed from pRSET vectors (Invitrogen, Carlsbad, CA) in E. coli BL21 (DE3) pLysS (Stratagene) and partially purified using nickel–nitrilotriacetic acid agarose resin (Qiagen). Inserts from pHT-ADK and pHT-ADKΔN13 were obtained by digestion with NcoI and HindIII and cloned into pRSET-B (Invitrogen) to generate pRSET-ADK and pRSET-ADKΔN13. SalI-HindIII and BamHI-HindIII fragments obtained from pRSET-ADK were inserted into pRSET-B to create pRSET-ADKΔN24 and pRSET-ADKΔN88, respectively. Expressed proteins were visualized by staining with Coomassie Brilliant Blue R 250 after polyacrylamide gel electrophoresis, and protein concentrations were estimated by comparing band intensities with BSA standards. Gel blot analysis was performed using an anti-His tag antibody. AL2 and L2 proteins fused to glutathione-S-transferase (GST-AL2 and GST-L2) likewise were expressed in BL21 cells and partially purified by glutathione-agarose chromatography. His-tagged AL2 (His-AL2) was expressed in insect (Sf9) cells using a baculovirus vector. Conditions for the expression and purification of the viral proteins have been described (Hartitz et al., 1999).

ADK Assays

Reactions were performed in a total volume of 15 μL and contained 50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 1 μM adenosine, and 5 μCi of γ-32P-ATP (3000 Ci/mmol) with 10 to 70 ng of ADK. After incubation at 37°C for 20 min, reactions were stopped by the addition of EDTA to 30 mM. Products were resolved by thin layer chromatography on polyethyleneimine-cellulose plates developed with 1 M acetic acid. To measure ADK activity in plants, stem pieces (3 mm) were obtained from transgenic Nicotiana benthamiana plants (AL2 line 472-1 and L2 line CTL2-6) (Sunter et al., 2001) and comparable nontransgenic plants at ∼4 weeks after germination. Crude extracts were obtained from 25 mg of stem tissue in 500 μL of 50 mM Hepes, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 5 mM DTT, 50 mM NaF, and 0.1 mM phenylmethylsulfonyl fluoride. Crude yeast extracts were obtained using the same buffer. Total protein concentration was estimated using the Bradford assay. In independent experiments, equal amounts of extracted protein from each sample (150 to 400 ng for plant experiments, 300 ng for yeast experiments) were added to the in vitro ADK assay. Labeled AMP generated was quantitated using a phosphorimager (Bio-Rad Molecular Imager FX, Hercules, CA). Extracts also were obtained from plants infected at 4 weeks after germination, and tissue was harvested at 14 days after inoculation. PVX and CMV were introduced by standard mechanical inoculation of infected sap. Plants were inoculated mechanically with TGMV DNA or agroinoculated with wild-type BCTV or BCTV L2 mutant virus as described previously (Sunter et al., 2001). Mock inocula consisted of buffer or Agrobacterium tumefaciens lacking viral DNA.

Immunolocalization of AL2

N. benthamiana plants were agroinoculated with TGMV or mock-inoculated with A. tumefaciens containing the binary vector alone as described previously (Sunter et al., 2001). Systemically infected leaf tissue from TGMV-inoculated plants, or comparable asymptomatic leaf tissue from mock-inoculated plants, was used for immunolocalization. Epidermis was peeled from the lower surface of several leaves, and the tissue was fixed in 4% paraformaldehyde in PBS and 0.1% Triton X-100 for 10 min on ice. Fixation was stopped with 3× PBS for 3 min on ice, followed by three washes in PBS and 0.1% Triton X-100. The tissue was stored in the same buffer for 3 h before immunolabeling.

Antibody labeling and washing steps were performed at 4°C. Fixed tissue was incubated with rabbit-derived primary antibody prepared against full-length TGMV AL2 protein (Cocalico, Reamstown, PA) at a 1:200 dilution in PBS overnight with shaking. After at least six washes with PBS and 0.1% Triton X-100 with shaking overnight, tissues were incubated overnight with shaking with fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG secondary antibody (1:1000 dilution in PBS and 0.1% Triton X-100). After at least six washes with PBS and 0.1% Triton X-100 for 6 h, the tissues were treated with 4′,6-diamidino-2-phenylindole (DAPI; 10 μg/mL) for nuclear staining. Tissue samples were examined with a fluorescence microscope (Axioskop; Carl Zeiss, Jena, Germany), and bright-field images were taken using differential interference contrast optics. FITC fluorescence was detected using a filter set with excitation at 485 nm and emission at 515 nm. DAPI fluorescence was detected using a filter set with excitation at 390 nm and emission at 460 nm.

Subcellular Localization of AL2

Native AL2 protein was expressed in insect cells using the Bac-to-Bac baculovirus expression system (Invitrogen) essentially as described (Hartitz et al., 1999). The intact TGMV AL2 open reading frame was obtained from pTGA26 (Sunter et al., 1990) by PCR and cloned into the baculovirus donor vector pFastBac. After selection, the recombinant bacmid containing the AL2 open reading frame (pTGA800) was transfected into Spodoptera frugiperda Sf9 cells. At 48 h after inoculation, infected cells were counted, harvested by centrifugation, and resuspended in nuclear isolation buffer (NIB; 10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT) at a rate of 10 × 106 cells/mL. Resuspended cells (100 × 106) were subjected to Dounce homogenization to release nuclei followed by centrifugation at 3300g to pellet nuclei. The supernatant was removed and subjected to centrifugation at 100,000g for 1 h to generate a soluble cytoplasmic protein fraction (S-100). Pelleted nuclei were washed with an equal volume of ice-cold 10% glycerol, centrifuged at 3300g, and resuspended in a final volume of 5 mL of 10% glycerol. Nuclei were purified further by centrifugation through a 2.1 M sucrose pad at 120,000g for 90 min. Purified nuclei were resuspended in 2 mL of NIB containing 10% glycerol and stored at −80°C.

Soluble cytoplasmic and nuclear fractions (equivalent to 5 × 105 cells) containing AL2 or chloramphenicol acetyltransferase as a control were electrophoresed through 4 to 20% Tris-glycine-SDS polyacrylamide gels (Invitrogen) in TGS buffer (25 mM Tris, 192 mM glycine, and 0.1% [w/v] SDS [Bio-Rad]). After electrophoresis, proteins were transferred to nitrocellulose, and native AL2 was detected by protein gel blot analysis using a primary antibody (1:10,000 dilution) directed against the full-length AL2 protein and a secondary alkaline phosphatase–conjugated goat anti-rabbit antibody (1:10,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega) were used for color development.

Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact D.M. Bisaro, bisaro.1@osu.edu

Accession Number

The GenBank accession number for the Arabidopsis genomic sequence containing ADK-C is AL162751.

Acknowledgments

We thank Janet Sunter for her tireless work to immunolocalize AL2 and Biao Ding and Yijun Qi for advice and assistance with the localization studies. We also are grateful to Erich Grotewold, Pappachan Kolattukudy, and Debbie Parris for comments and critical reading of the manuscript and to Jim Carrington for helpful discussion. We thank Stephen Elledge for providing yeast two-hybrid materials, including plasmids pAS2 and pACT2 and yeast strain Y190, and Paul Herman for providing yeast expression plasmids YEpL and YEpU. We also thank Per Nygaard for providing E. coli HO4 and Gregory Phillips for pDHK29/30. Deoxycoformycin was a gift from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (National Institutes of Health, Bethesda, MD). The Arabidopsis cDNA library was obtained from the ABRC at Ohio State University (National Science Foundation/Department of Energy/U.S. Department of Agriculture Collaborative Research in Plant Biology Program, Research Collaboration Group in Plant Protein Phosphorylation [USDA 92-37105-7675]). This work was supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015180.

References

  1. Ahlquist, P. (2002). RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 296, 1270–1273. [DOI] [PubMed] [Google Scholar]
  2. Bass, B. (2000). Double-stranded RNA as a template for gene silencing. Cell 101, 235–238. [DOI] [PubMed] [Google Scholar]
  3. Baulcombe, D.C. (1999). Fast forward genetics based on virus-induced gene silencing. Curr. Opin. Plant Biol. 2, 109–113. [DOI] [PubMed] [Google Scholar]
  4. Bender, J. (2001). A vicious cycle: RNA silencing and DNA methylation in plants. Cell 106, 129–132. [DOI] [PubMed] [Google Scholar]
  5. Bisaro, D.M. (1996). Geminivirus replication. In DNA Replication in Eukaryotic Cells, M. DePamphilis, ed (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 833–854.
  6. Brigneti, G., Voinnet, O., Li, W.-X., Ji, L.-H., Ding, S.-W., and Baulcombe, D.C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739–6746. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  7. Carrington, J.C., Kasschau, K.D., and Johansen, L.K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1–5. [DOI] [PubMed] [Google Scholar]
  8. Davies, S.P., Helps, N.R., Cohen, P.T.W., and Hardie, D.G. (1995). 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase: Studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2Ac. FEBS Lett. 377, 421–425. [DOI] [PubMed] [Google Scholar]
  9. Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A.E., Lee, W.-H., and Elledge, S.J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555–569. [DOI] [PubMed] [Google Scholar]
  10. Gutierrez, C. (1999). Geminivirus DNA replication. Cell. Mol. Life Sci. 56, 313–329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halford, N., and Hardie, D.G. (1998). SNF1-related protein kinases: Global regulators of carbon metabolism in plants? Plant Mol. Biol. 37, 735–748. [DOI] [PubMed] [Google Scholar]
  12. Hanley-Bowdoin, L., Settlage, S., Orozco, B.M., Nagar, S., and Robertson, D. (1999). Geminiviruses: Models for plant DNA replication, transcription, and cell cycle regulation. Crit. Rev. Plant Sci. 18, 71–106. [PubMed] [Google Scholar]
  13. Hao, L., Wang, H., Sunter, G., and Bisaro, D.M. (2003). Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15, 1034–1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hardie, D.G., Carling, D., and Carlson, M. (1998). The AMP-activated/SNF1 protein kinase subfamily: Metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem. 67, 821–855. [DOI] [PubMed] [Google Scholar]
  15. Hardie, D.G., and Hawley, S.A. (2001). AMP-activated protein kinase: The energy charge hypothesis revisited. Bioessays 23, 1112–1119. [DOI] [PubMed] [Google Scholar]
  16. Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., and Elledge, S.J. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinase. Cell 75, 805–816. [DOI] [PubMed] [Google Scholar]
  17. Hartitz, M.D., Sunter, G., and Bisaro, D.M. (1999). The geminivirus transactivator (TrAP) is a single-stranded DNA and zinc-binding phosphoprotein with an acidic activation domain. Virology 263, 1–14. [DOI] [PubMed] [Google Scholar]
  18. Hawley, S.A., Selbert, M.A., Goldstein, E.G., Edelman, A.M., Carling, D., and Hardie, D.G. (1995). 5′-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J. Biol. Chem. 270, 27186–27191. [DOI] [PubMed] [Google Scholar]
  19. Hormuzdi, S.G., and Bisaro, D.M. (1995). Genetic analysis of beet curly top virus: Examination of the roles of L2 and L3 genes in viral pathogenesis. Virology 206, 1044–1054. [DOI] [PubMed] [Google Scholar]
  20. Hove-Jensen, B., and Nygaard, P. (1989). Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli. J. Gen. Microbiol. 135, 1263–1273. [DOI] [PubMed] [Google Scholar]
  21. Jackson, J.P., Lindroth, A.M., Cao, X., and Jacobsen, S.E. (2002). Control of CpNpG methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556–560. [DOI] [PubMed] [Google Scholar]
  22. Johnston, M. (1999). Feasting, fasting, and fermenting: Glucose sensing in yeast and other cells. Trends Genet. 15, 29–33. [DOI] [PubMed] [Google Scholar]
  23. Jones, L., Ratcliff, F., and Baulcombe, D.C. (2001). RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Curr. Biol. 11, 747–757. [DOI] [PubMed] [Google Scholar]
  24. Kasschau, K.D., and Carrington, J.C. (1998). A counterdefensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell 95, 461–470. [DOI] [PubMed] [Google Scholar]
  25. Kjemtrup, S., Sampson, K.S., Peele, C.G., Nguyen, L.V., Conkling, M.A., Thompson, W.F., and Robertson, D. (1998). Gene silencing from plant DNA carried by a geminivirus. Plant J. 14, 91–100. [DOI] [PubMed] [Google Scholar]
  26. Lecoq, K., Belloc, I., Desgranges, C., and Daignan-Fornier, B. (2001). Role of adenosine kinase in Saccharomyces cerevisiae: Identification of the ADO1 gene and study of the mutant phenotypes. Yeast 18, 335–342. [DOI] [PubMed] [Google Scholar]
  27. Moffatt, B.A., Stevens, Y.Y., Allen, M.S., Snider, J.D., Periera, L.A., Todorova, M.I., Summers, P.S., Weretilnyk, E.A., Martin-Caffrey, L., and Wagner, C. (2002). Adenosine kinase deficiency is associated with developmental abnormalities and reduced transmethylation. Plant Physiol. 128, 812–821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moffatt, B.A., Wang, L., Allen, M.S., Stevens, Y.Y., Qin, W., Snider, J., and von Schwartzenberg, K. (2000). Adenosine kinase of Arabidopsis: Kinetic properties and gene expression. Plant Physiol. 124, 1775–1785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paszkowski, J., and Whitham, S.A. (2001). Gene silencing and DNA methylation processes. Curr. Opin. Plant Biol. 4, 123–129. [DOI] [PubMed] [Google Scholar]
  30. Phillips, G.J., Park, S.-K., and Huber, D. (2000). High copy number plasmids compatible with commonly used cloning vectors. Biotechniques 28, 400–408. [DOI] [PubMed] [Google Scholar]
  31. Pilartz, M., and Jeske, H. (1992). Abutilon mosaic virus double-stranded DNA is packed into minichromosomes. Virology 189, 800–802. [DOI] [PubMed] [Google Scholar]
  32. Plasterk, R.H.A. (2002). RNA silencing: The genome's immune system. Science 296, 1263–1265. [DOI] [PubMed] [Google Scholar]
  33. Sharp, P.A. (2001). RNA interference: 2001. Genes Dev. 15, 485–490. [DOI] [PubMed] [Google Scholar]
  34. Stanley, J., Latham, J., Pinner, M.S., Bedford, I., and Markham, P.G. (1992). Mutational analysis of the monopartite geminivirus beet curly top virus. Virology 191, 396–405. [DOI] [PubMed] [Google Scholar]
  35. Sugden, C., Crawford, R.M., Halford, N.G., and Hardie, D.G. (1999. a). Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5′-AMP. Plant J. 19, 433–439. [DOI] [PubMed] [Google Scholar]
  36. Sugden, C., Donaghy, P.G., Halford, N., and Hardie, D.G. (1999. b). Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxyl-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro. Plant Physiol. 120, 257–274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sunter, G., and Bisaro, D.M. (1992). Transactivation of geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription. Plant Cell 4, 1321–1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sunter, G., and Bisaro, D.M. (1997). Regulation of a geminivirus coat protein promoter by AL2 protein (TrAP): Evidence for activation and derepression mechanisms. Virology 232, 269–280. [DOI] [PubMed] [Google Scholar]
  39. Sunter, G., and Bisaro, D.M. (2003). Identification of a minimal sequence required for activation of the tomato golden mosaic virus coat protein promoter in protoplasts. Virology 305, 452–462. [DOI] [PubMed] [Google Scholar]
  40. Sunter, G., Hartitz, M.D., Hormuzdi, S.G., Brough, C.L., and Bisaro, D.M. (1990). Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication. Virology 179, 69–77. [DOI] [PubMed] [Google Scholar]
  41. Sunter, G., Sunter, J., and Bisaro, D.M. (2001). Plants expressing tomato golden mosaic virus AL2 or beet curly top virus L2 transgenes show enhanced susceptibility to infection by DNA and RNA viruses. Virology 285, 59–70. [DOI] [PubMed] [Google Scholar]
  42. Tang, G., Reinhart, B.J., Bartel, D.P., and Zamore, P.D. (2003). A biochemical framework for RNA silencing in plants. Genes Dev. 17, 49–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vance, V., and Vaucheret, H. (2001). RNA silencing in plants: Defense and counterdefense. Science 292, 2277–2280. [DOI] [PubMed] [Google Scholar]
  44. van Wezel, R., Liu, H., Tien, P., Stanley, J., and Hong, Y. (2001). Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus. Mol. Plant-Microbe Interact. 14, 1125–1128. [DOI] [PubMed] [Google Scholar]
  45. van Wezel, R., Liu, H., Tien, P., Stanley, J., and Hong, Y. (2002). Mutation of three cysteine residues in tomato yellow leaf curl virus-China C2 protein causes dysfunction in pathogenesis and posttranscriptional gene silencing-suppression. Mol. Plant-Microbe Interact. 15, 203–208. [DOI] [PubMed] [Google Scholar]
  46. Voinnet, O., Pinto, Y.M., and Baulcombe, D.C. (1999). Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96, 14147–14152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I.S., and Martienssen, R.A. (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837. [DOI] [PubMed] [Google Scholar]
  48. von Schwartzenberg, K., Kruse, S., Reski, R., Moffatt, B., and Laloue, M. (1998). Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism. Plant J. 13, 249–257. [DOI] [PubMed] [Google Scholar]
  49. Waterhouse, P.M., Wang, M.B., and Lough, T. (2001). Gene silencing as an adaptive defense against viruses. Nature 411, 834–842. [DOI] [PubMed] [Google Scholar]
  50. Weretilnyk, E.A., Alexander, K.J., Drebenstedt, M., Snider, J.D., Summers, P.S., and Moffatt, B.A. (2001). Maintaining methylation activities during salt stress: The involvement of adenosine kinase. Plant Physiol. 125, 856–865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zamore, P.D. (2002). Ancient pathways programmed by small RNAs. Science 296, 1265–1269. [DOI] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES