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First published online October 13, 2006; 10.1104/pp.106.088476 Plant Physiology 142:1642-1655 (2006) © 2006 American Society of Plant Biologists Geminivirus Infection Up-Regulates the Expression of Two Arabidopsis Protein Kinases Related to Yeast SNF1- and Mammalian AMPK-Activating Kinases1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695–7622
Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid studies. Using specific antibodies, we showed that both Arabidopsis kinases are elevated in infected leaves. Immunoblot analysis of healthy plants revealed that GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or undetectable in mature tissues. Immunohistochemical staining showed that the kinases accumulate in the shoot apical meristem, leaf primordium, and emerging petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show a small increase during infection and do not change significantly during development. Treating healthy seedlings and infected leaves with the proteasome inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas treatment with the translation inhibitor cycloheximide reduced both kinases, demonstrating that their accumulation is modulated by posttranscriptional processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate the yeast kinase SNF1 and its mammalian homolog AMPK, respectively. Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1 and GRIK2 can functionally replace the yeast kinases, suggesting that the Arabidopsis kinases mediate one or more processes during early plant development and geminivirus infection by activating SNF1-related kinases.
Protein kinases are important components of signal transduction and regulatory pathways in all eukaryotes. In plants, they play critical roles in pathogen recognition and the defense response (for review, see Romeis, 2001  ; Xing et al., 2002; Pedley and Martin, 2003, 2005). They can also act in concert with pathogen proteins to facilitate infection (Florentino et al., 2006). This partnership is particularly important for viruses that have limited coding capacities. Geminiviruses are small DNA viruses that depend on host machinery to transcribe and replicate their genomes in plant cells (for review, see Hanley-Bowdoin et al., 2004). These important plant pathogens also interact with host factors to move throughout the plant and to overcome host defenses. Host protein kinases have been implicated in these processes through their interactions with geminivirus proteins. Analysis of these interactions will increase our understanding of the mechanisms leading to productive geminivirus infection and provide valuable insight into the roles of these kinases in plant growth and development.
Geminiviruses are a large family of plant-infecting viruses that fall into four genera based on their genome structure, insect vector, and host range (Rojas et al., 2005
Geminivirus proteins interact with a diverse set of host factors. Many of these interactions have been implicated in the recruitment of plant proteins to participate in essential viral processes. AL1 and AL3 recruit host replication machinery by binding to PCNA and RFC, the clamp and clamp loader of the host DNA polymerase
Binding of geminivirus proteins can also sequester and/or inhibit the activities of plant proteins to overcome barriers to infection or host defense mechanisms. AL1 and AL3 interact with the retinoblastoma-related protein (pRBR), a negative regulator of cell cycle progression and a differentiation factor (Weinberg, 1995
In plants, receptor-like protein kinases (RLKs) are often associated with the plasma membrane and activated by direct interaction with intracellular or extracellular signal molecules (for review, see Morris and Walker, 2003
A Small Family of Arabidopsis Protein Kinases Interact with a Geminivirus Replication Protein
In an earlier study (Kong and Hanley-Bowdoin, 2002
GRIK1 and GRIK2 are 396 and 407 residues in length, respectively, and share 77% amino acid identity and 85% similarity overall (Fig. 1). The GRIK1 kinase domain consists of 262 amino acids, whereas the GRIK2 kinase domain has 264 residues. The domains, which are 88% identical and 93% similar, contain the ATP-binding and catalytic motifs characteristic of Ser/Thr protein kinases (Hanks and Hunter, 1995 ). The GRIK1 and GRIK2 kinase domains are flanked by N-terminal sequences of 106 and 105 amino acids and C-terminal sequences of 27 and 41 residues, respectively. The N-terminal domains are 57% identical and 71% similar, while the C-terminal sequences are less conserved with 46% identity and 65% similarity. BLAST analysis of plant databases uncovered two protein kinase sequences that are related to the Arabidopsis kinases (Fig. 1). The rice (Oryza sativa) Os03g50330 gene (The Institute for Genomic Research nomenclature) encodes an expressed protein that is equally related to GRIK1 (54% identity and 65% similarity) and GRIK2 (52% identity and 64% similarity). The Medicago truncatula bacterial artificial chromosome mth2-22g6 specifies a predicted protein (GenBank ABE79890) that is also equally related to GRIK1 (60% identity and 72% similarity) and GRIK2 (59% identity and 72% similarity). Like the Arabidopsis kinases, the rice and M. truncatula proteins contain highly conserved, central kinase domains flanked by more divergent N- and C-terminal sequences. The M. truncatula protein contains a 15-amino acid sequence in the kinase domain that is absent in the other sequences. The rice protein has two insertions in the N-terminal domain of 21 and six amino acids compared to the dicot proteins. Taken together, these proteins constitute a small protein kinase family in plants.
We asked if full-length GRIK1 and GRIK2 bind to TGMV AL1 in yeast two-hybrid assays as shown previously for the truncated GRIK protein (Kong and Hanley-Bowdoin, 2002
Peptide Antibodies against GRIK1 and GRIK2 To distinguish between the GRIK1 and GRIK2 proteins, we took advantage of the divergence in their C-terminal sequences to generate unique peptides for antibody production (Fig. 1). The peptide antibodies were used in immunoblotting experiments of total protein extracts from insect cells expressing either His-tagged GRIK1 or GRIK2. The anti-GRIK1 antiserum detected an approximately 50-kD protein in cells expressing recombinant His-GRIK1 (Fig. 3A , lane 1) but not in cells expressing His-GRIK2 (lane 2). Conversely, the anti-GRIK2 antiserum did not cross-react with protein in cells expressing recombinant His-GRIK1 (Fig. 3A, lane 3) and, instead, detected a protein of expected size in cells expressing His-GRIK2 (lane 4). Together, these results show that peptide antibodies are specific for GRIK1 or GRIK2.
The relative sensitivities of the peptide antibodies were compared using the recombinant His-tagged kinases. An anti-His-tag antibody was used to determine the volumes of insect cell protein extracts that gave similar signals for His-GRIK1 and His-GRIK2 on immunoblots (Fig. 3B, compare lanes 1 and 2). Based on this comparison, one volume of the GRIK1 extract and 1/40 volume of the GRIK2 extract produced bands of similar intensity with their respective peptide antibodies (Fig. 3B, compare lanes 3 and 4). These data established that the GRIK2 antibody is approximately 40-fold more sensitive relative to the GRIK1 antibody in our experimental conditions.
We asked if the affinity-purified peptide antibodies could detect GRIK1 and GRIK2 proteins in total protein extracts from symptomatic Arabidopsis leaves at 12 d postinoculation (dpi) with Cabbage leaf curl virus (CaLCuV), a member of the Begomovirus genus. Bands of the expected size were seen on immunoblots probed with the GRIK1 (Fig. 4A , lane 2) or GRIK2 (lane 4) antibodies. Lower levels of both kinases were detected in extracts from mock-inoculated leaves (Fig. 4A, lanes 1 and 3), demonstrating that their accumulation increases during CaLCuV infection. A polyclonal antibody against the CaLCuV AL1 protein detected the viral replication protein in extracts from infected leaves (Fig. 4A, lane 6) but not from mock-inoculated leaves (lane 5).
We also examined GRIK1 and GRIK2 accumulation in Arabidopsis leaves infected with Beet curly top virus (BCTV Logan), a member of the Curtovirus genus. Because BCTV-inoculated Arabidopsis plants are asymptomatic at 12 dpi, we isolated protein extracts from symptomatic and mock-inoculated leaves at 28 dpi for immunoblot analysis with the peptide antibodies. Higher levels of both kinases were detected in BCTV-infected leaves (Fig. 4B, lanes 2 and 4) than in mock-inoculated leaves (lanes 1 and 3). We were unable to monitor the BCTV replication protein C1 in parallel because our antibodies against begomovirus AL1 proteins do not cross-react with the curtovirus protein. However, the results demonstrated that diverse geminiviruses stimulate the accumulation of the GRIK1 and GRIK2 proteins during infection.
We investigated if increases in the steady-state levels of GRIK1 and GRIK2 mRNAs during infection are responsible for the higher protein levels in Arabidopsis leaves. Relative mRNA levels were measured in three independent experiments by real-time quantitative reverse transcription (RT)-PCR using gene-specific primers for GRIK1, GRIK2, and the Act2 reference gene. Higher transcript levels were detected for both kinase genes in CaLCuV-infected leaves versus mock-inoculated leaves at 12 dpi, with increases of 2.1-fold and 1.6-fold for GRIK1 and GRIK2 mRNA, respectively (Fig. 4C). Although the increases in GRIK1 and GRIK2 mRNA levels in infected leaves were statistically significant in Z tests (P < 0.05), they were not sufficient to account for the higher protein levels during infection (Fig. 4A).
The RNA analysis suggested that the accumulation of GRIK1 and GRIK2 proteins is modulated at least in part by posttranscriptional processes. Protein degradation through the ubiquitin-proteasome pathway is a common mechanism for rapid and effective control of cellular levels of regulatory proteins like protein kinases (Sullivan et al., 2003
Consistent with the seedling results, there were slight increases in the GRIK1 and GRIK2 levels 3 h after MG132 treatment in mock-inoculated leaves (Fig. 5B, lane 2) compared to the solvent-only control (lane 1). However, MG132 treatment of infected leaves (Fig. 5B, lane 4) resulted in substantial increases in GRIK1 and GRIK2 abundance compared to the solvent treatment (lane 3). The similar levels of GRIK1 and GRIK2 in mock and infected leaves treated with solvent only (Fig. 5B, compare lanes 1 and 3) are consistent with degradation of the kinases during the 3-h treatment in the absence of the inhibitor. Higher levels of the AL1 protein were also observed in the presence of MG132 (Fig. 5B, lanes 3 and 4), but the difference between MG132 and solvent-treated samples was less than seen for the kinases. These results indicated that GRIK1 and GRIK2 are degraded via the ubiquitin-proteasome pathway in CaLCuV-infected plant leaves. The significantly higher levels of GRIK1 and GRIK2 in CaLCuV-infected leaves versus mock-inoculated leaves in the MG132-treated samples (Fig. 5B, compare lanes 2 and 4) also suggested that the synthesis of both kinases is elevated during infection. We used the protein synthesis inhibitor cycloheximide to confirm that de novo protein synthesis is required to maintain GRIK1 and GRIK2 protein levels during infection. GRIK1 levels were reduced and GRIK2 was not detected in infected leaves incubated with cycloheximide for 3 h prior to protein extraction (Fig. 5C, lane 2) compared to solvent-treated tissue (lane 1). In contrast, AL1 protein levels were not impacted by cycloheximide treatment (Fig. 5C, compare lanes 1 and 2). In parallel assays, high levels of GRIK1 and GRIK2 were detected in the presence of MG132 (Fig. 5C, lane 3). Together, these results indicate that protein synthesis and turnover influence GRIK1 and GRIK2 protein levels during infection.
AL1 interacts with the host pRBR to induce the expression of DNA replication machinery and cause mature plant cells to reenter the S phase of cell cycle (Ach et al., 1997
The relative amounts of GRIK1 and GRIK2 in young leaves were compared using different amounts of a total protein extract and their peptide antibodies in Figure 3B. Bands of similar intensities were seen for the two kinases when 25 µg of total plant protein was probed with the GRIK1 antibody and 10 µg of total protein was probed with the GRIK2 antibody (Fig. 3B, compare lanes 5 and 6). Given that the GRIK2 antibody displays approximately 40-fold greater sensitivity than the GRIK1 antibody under these experimental conditions (Fig. 3B, lanes 3 and 4), the amount of GRIK1 protein in young leaves is approximately 10-fold higher than the GRIK2 protein. The steady-state mRNA levels corresponding to the kinase genes were measured by real-time quantitative RT-PCR in the four leaf samples in two independent experiments. For both GRIK1 and GRIK2, there was no statistically significant difference in relative mRNA abundance between any of the samples (Fig. 6B). These data are consistent with available microarray data showing that GRIK1 and GRIK2 mRNAs levels do not vary in a range of tissue types and developmental stages (http://www.arabidopsis.org) and suggest that their protein levels are posttranscriptionally regulated in developing, mature, and senescent leaves. GRIK1 and GRIK2 expression in other Arabidopsis organs was examined by immunoblotting with the peptide antibodies. Both kinases were detected in flower buds (Fig. 6C, lanes 1 and 5), fully opened flowers (lanes 2 and 6), and siliques (lanes 3 and 7), but neither was present in roots (lanes 4 and 8). The GRIK2 protein level was higher in flower buds and siliques than in mature flowers (Fig. 6C, lanes 5 and 7 with lane 6), further supporting the idea that the GRIK proteins are associated with young plant tissues that contain dividing and endoreduplicating cells.
We compared the cellular and subcellular distributions of GRIK1 and GRIK2 in 5-week-old Arabidopsis plants using the peptide antibodies for immunolocalization. These studies focused on the SAM, leaf primordia, and emerging petioles of leaves immediately flanking the primordium, because the kinases are the predominant immunoreactive proteins in these tissues (Fig. 6A, lanes 1 and 5). In addition, these tissues contain few or no plastids, which were also a source of potential background. Both peptide antibodies stained chloroplasts in mature leaves (not shown) even though neither kinase protein was detected in this tissue by immunoblotting (Fig. 6A, lanes 3 and 7).
The GRIK1 antibody stained a variety of cells in the SAM (Fig. 7D
), leaf primordium (Fig. 7E), and petiole (Fig. 7F). The stained cells included cells in the SAM central zone, epidermal and emerging mesophyll cells in the leaf primordium, and epidermal cells in the petiole. The strongest GRIK1 staining colocalized with 4',6-diamidino-2-phenylindole staining (data not shown), indicating that the kinase is in the nucleus. Cytoplasmic GRIK1 staining was also visible in the SAM (Fig. 7D) but was not readily apparent in the leaf primordium or petiole sections when compared to the corresponding normal rabbit antibody controls (compare Fig. 7, B and E, with C and F). These data are consistent with earlier results using a GRIK polyclonal antibody showing that the kinase is in nuclei of developing leaf cells of Nicotiana benthamiana plants (Kong and Hanley-Bowdoin, 2002
In contrast, GRIK2 antibody staining was weak in all three tissues (Fig. 7, G–I). This was unexpected because of the greater sensitivity of the antibody on immunoblots. The weak staining could not be attributed to the 20-kD cross-reacting band detected on immunoblots because the same band was observed in young and mature leaf extracts (Fig. 6A, compare lanes 5 and 7), but mature leaves did not show staining in parallel assays (data not shown). Instead, the weak staining may reflect the distribution of GRIK2 throughout the cell, making it more difficult to detect. Consistent with this idea, there appears to be more general GRIK2 staining in all three tissues compared to the normal rabbit antibody controls (compare Fig. 7, A and G; B and H; and C and I). However, a few stained nuclei can be seen in the leaf primordium and petiole sections (Fig. 7, H and I), indicating that GRIK2 is not excluded from the nucleus. Another possibility is that the GRIK2 C terminus is masked and unavailable for antibody binding in the native protein in the fixed tissue sections. In either case, the distinct staining patterns for GRIK1 and GRIK2 are indicative of differences in their local concentrations and/or differential localization.
GRIK1 and GRIK2 form a small protein kinase family in Arabidopsis and are distinct from their next most similar protein kinase, AtKIN10, a SnRK1 family protein kinase. BLAST searches of the Saccharomyces cerevisiae and human proteomes revealed that GRIK1 and GRIK2 are most similar to a family of protein kinases that activate the yeast SNF1 kinase or its mammalian homolog AMPK (for review, see Witters et al., 2006
In yeast, the PAK1/TOS3/ELM1 kinases phosphorylate and activate the downstream kinase SNF1, which facilitates utilization of non-Glc carbon sources (Hong et al., 2003 ). Human CaMKK / activate their substrate AMPK, which is homologous to yeast SNF1 (Hawley et al., 2005; Hong et al., 2005; Hurley et al., 2005). Both CaMKK and CaMKK can complement a yeast triple mutant disrupted in the PAK1/TOS3/ELM1 genes, which regains the capacity to grow on medium containing non-Glc carbon sources in the presence of the human kinases (Hong et al., 2005). We asked if Arabidopsis GRIK1 or GRIK2 is also able to functionally replace the yeast PAK1/TOS3/ELM1 kinases. The triple mutant strain MCY5138 (Hong et al., 2003) was transformed with expression cassettes corresponding to GRIK1, GRIK2, or their mutant forms, GRIK1(K136A) and GRIK2(K137A) (Fig. 1), under the control of the yeast ADH1 gene promoter. Immunoblotting of protein extracts from transformants grown in Glc-containing medium indicated that GRIK1 (Fig. 8B, lane 2), GRIK1(K136A) (lane 3), GRIK2 (lane 4), and GRIK2(K137A) (lane 5) were expressed in the pak1/tos3/elm1 mutant. We then plated a dilution series for each transformant on synthetic complete media containing Glc, raffinose, or glycerol-ethanol as the carbon source. MCY5138 cells transformed with an empty expression cassette served as the negative control. All of the transformants showed similar growth on Glc medium (Fig. 8C, top). On raffinose medium, weak growth was seen only at lower dilutions for the transformants carrying the empty vector, the GRIK1(K136A) cassette, or the GRIK2(K137A), while both GRIK1 and GRIK2 transformants grew well at all the dilutions tested (Fig. 8C, middle). On glycerol-ethanol medium, growth was only detected for the GRIK2 transformant, which grew at all dilutions tested (Fig. 8C, bottom). These data demonstrated that GRIK1 or GRIK2 can complement the pak1/tos3/elm1 mutations in yeast and that complementation depends on their kinase activities. They also suggest that Arabidopsis GRIK1 and GRIK2 mediate similar biochemical functions to yeast PAK1/TOS3/ELM1 and mammalian CaMKK/. However, GRIK2 complementation was stronger than GRIK1, indicating that the Arabidopsis kinases may not be functionally equivalent.
Geminiviruses have coevolved with their plant hosts to use endogenous cellular processes to replicate, express, and transport their DNA genomes and to overcome resistance mechanisms (Rojas et al., 2005 ). This is achieved in part by modulating existing signal transduction pathways through the interaction of viral proteins and host protein kinases. Geminivirus proteins bind to RLKs that are part of the plant pathogen surveillance system and interfere with the ability of the host to mount a resistance response (Fontes et al., 2004). Susceptibility to geminivirus infection is also enhanced by interaction with an SnRK1, implicating this evolutionarily conserved, energy-sensing protein kinase in the plant defense response (Hao et al., 2003). In this article, we provide another connection between SnRK1 and geminivirus infection by showing that the AL1 partners GRIK1 and GRIK2 can act as SNF1-activating kinases in yeast. Data showing that the GRIK1 and GRIK2 proteins accumulate preferentially during infection and in young Arabidopsis tissues suggest that the SnRK1 cascade regulates a diverse range of functions in higher plants. The Arabidopsis genome encodes two related protein kinases, GRIK1 and GRIK2, which are the sole members of the kinase family 4.2.7 as defined by the PlantsP Kinase Classification Scheme (http://plantsp.genomics.purdue.edu). Related kinases are encoded by the rice and M. truncatula genomes (Fig. 1), demonstrating that the 4.2.7 family is conserved in both monocot and dicot plant species. The GRIK1 and GRIK2 coding sequences display 82% nucleotide identity, and their genes are arranged similarly with respect to exon-intron location and exon size, characteristic of a gene duplication event. GRIK1 and GRIK2 also have many attributes in common, including their expression patterns, posttranslational regulation, interaction with a geminivirus replication protein, and complementation of the yeast pak1/tos3/elm1 triple mutant. These similarities suggest that GRIK1 and GRIK2 are functionally redundant, but two observations argue against this conclusion. Immunolocalization studies suggested that they are in different cellular compartments or associated with different protein complexes (Fig. 7). In addition, GRIK2 displayed a stronger complementation phenotype in the yeast triple mutant (Fig. 8C). This was most striking when yeast was grown in the presence of glycerol and ethanol, with only GRIK2 showing complementation.
GRIK1 and GRIK2 accumulation is associated with young leaf tissues (Fig. 6A) and induced in older leaves by geminivirus infection (Fig. 4, A and B). Immunolocalization studies showed that the kinases are abundant in all cell types of the SAM, leaf primordium, and emerging petiole. Cells at these early developmental stages and geminivirus-infected cells share the capacity to replicate DNA either as part of a mitotic cell cycle or an endocycle (Traas et al., 1998
Several lines of evidence indicated that GRIK1 and GRIK2 expression is regulated by multiple mechanisms during geminivirus infection. GRIK1 and GRIK2 mRNAs are elevated approximately 2-fold in infected leaves (Fig. 4C), but this change is not sufficient to account for the much larger rise in protein levels (Fig. 4A), further indicating that posttranscriptional mechanisms contribute to the accumulation of the kinases. Consistent with this idea, MG132 treatment of CaLCuV-infected leaves established that GRIK1 and GRIK2 are degraded via the proteasome pathway and uncovered a large increase in de novo protein synthesis during infection (Fig. 5, B and C). The net accumulation of GRIK1 and GRIK2 most likely reflects a balance between these two processes. Interestingly, the 5'-untranslated regions of both GRIK1 and GRIK2 mRNAs contain multiple upstream small open reading frames (data not shown), which have been associated with translational regulation of other mRNAs (Morris and Geballe, 2000
Phylogenetic analysis showed that GRIK1 and GRIK2 are related to the S. cerevisiae kinases PAK1, TOS3, and ELM1 and to human CaMKKs. This analysis also uncovered related kinases in Schizosaccharomyces pombe, Dictyostelium discoideum, Caenorhabditis elegans, zebrafish (Danio rerio), and Drosophila melanogaster, indicating that GRIK1 and GRIK2 belong to a kinase family that is conserved across eukaryotes. This family branch in the phylogenetic tree is supported by a bootstrap value of 74%, which increases to 84% if the more diverse ELM1 is not considered (Fig. 8A). These data agree with an earlier study comparing protein kinases from Arabidopsis and budding yeast that did not conclusively identify GRIK1 and GRIK2 as homologs of PAK1/TOS3/ELM1 (Wang et al., 2003a
The Arabidopsis SnRK family has 38 members constituting three subfamilies (Hrabak et al., 2003
Geminiviruses modify differentiation and cell cycle controls in mature leaf cells to induce replication of host chromosomal DNA as well as their own genomes (Nagar et al., 2002
GRIK1 and GRIK2 might also contribute to the host defense response by up-regulating the SnRK1 pathway and conferring reduced susceptibility to geminivirus infection (Hao et al., 2003
The potential involvement of GRIK1 and GRIK2 in the defense response seems counter to the hypothesis that the kinases facilitate infection by activating SnRK-regulated nutrient metabolism. However, these possibilities may not be mutually exclusive in a dynamic, ordered process in which AL1 and AL3 expression and GRIK accumulation precedes AL2 expression and inhibition of a SnRK cascade. Early in the infection process, the viral and host replication components are put into place, with GRIK1 and GRIK2 activation of the SnRK cascade ensuring that the requisite precursors and energy sources are available for virus propagation. Later, production of the AL2 protein counters the capacity of activated SnRK1 to also interfere with the infection process. This fine temporal control could have evolved during the long evolutionary relationship between geminiviruses and their hosts as a way to ensure viral propagation and plant survival (Rojas et al., 2005
Plant Growth and Treatments Arabidopsis (Arabidopsis thaliana) Columbia-0 plants were grown at 20°C in a Percival reach-in chamber with 8/16-h light/dark cycles under a light intensity of 15,000 Lux. Leaves from 5-week-old rosette plants or flower buds, fully open flowers, and siliques from bolted plants grown in soil were collected for protein and RNA extractions. Young seedlings were grown on petri dishes containing 1x Murashige and Skoog salts, 1x Gamborg's B5 vitamins, 1% Suc, and 0.7% agar for 2 weeks. The seedling roots were collected for protein extraction, or whole seedlings were removed and subjected to MG132 treatment before extraction.
Five-week-old, soil-grown plants were infected in the apex by syringe inoculation of Agrobacterium tumefaciens carrying pNSB1090 and pNSB1091, which contain partial tandem copies of CaLCuV A and B DNA, respectively (Egelkrout et al., 2002 For treatment with MG132, plant materials were submerged for 3 h in 1x Murashige and Skoog salts solution containing 50 µM MG132 previously dissolved in dimethyl sulfoxide. The equivalent volume of dimethyl sulfoxide in 1x Murashige and Skoog salts was used as the control treatment. Plant tissues were treated similarly with 100 µM cycloheximide except that ethanol was used as the solvent.
To obtain the full-length cDNA for GRIK1, Arabidopsis leaf RNAs were reverse transcribed using the primer 5'-TATGAGCATGAAGGTACATGAG-3'. The cDNA was amplified using the same primer and the second primer, 5'-GGCTCGAGGATCCGATGTTTTGTGATAGT-3'. The DNA fragment corresponding to the 5'-end of the GRIK1 coding sequence was digested with BamHI and DraIII and used to replace the BamHI-DraIII fragment of pNSB1016, a yeast two-hybrid prey plasmid that specifies GRIK1 residues 12 to 396 (Kong and Hanley-Bowdoin, 2002
The cDNA for full-length GRIK2 was cloned by RT-PCR using the primer 5'-GGAGCTCGAATTCTTAGTTAGGATCTGAGGTTTC-3' for both reactions and the primer 5'-GGAACGCCATATGTTTCGTGATAGTTTTTTGTTTGC-3' for PCR. The amplification product was digested with NdeI and EcoRI and subcloned into the same sites of pNSB910 (Castillo et al., 2004
The insect transfer vector pNSB1184 was used to make a recombinant baculovirus corresponding to His-tagged GRIK2. The transfer vector for His-tagged GRIK1(12–396) was described previously (Kong and Hanley-Bowdoin, 2002
Two peptides, CKRKAEEEEDQNHS and SKIEEGEANGISETS, located at the C termini of GRIK1 and GRIK2, respectively, were synthesized and conjugated to keyhole limpet hemocyanin for immunizing rabbits (Washington Biotechnology). Specific antibodies were affinity purified with the GRIK1 and GRIK2 peptides coupled to the SulfoLink Coupling Gel (Pierce) and Affi-Gel 102 (Bio-Rad) resins, respectively, according to the manufacturers' instructions. The antibodies were purified further by passage through keyhole limpet hemocyanin-agarose (Sigma). Antiserum against CaLCuV AL1 was prepared by immunizing rabbits with His-tagged recombinant protein produced in Escherichia coli (J.T. Ascencio-Ibañez and L. Hanley-Bowdoin, unpublished data). Monoclonal antibodies against the His tag were from CLONTECH.
Proteins used in this study were from total cell lysates. Plant tissues were collected and ground into fine powders in liquid nitrogen, suspended in 1 volume of 2x SDS-PAGE sample buffer, boiled for 15 min, and centrifuged at 16,000g for 10 min to remove cell debris. Attached insect cells expressing recombinant proteins were solubilized with 1x SDS-PAGE sample buffer, boiled, and centrifuged in the same way. Yeast (Saccharomyces cerevisiae) cells from a 5-mL overnight culture in liquid synthetic medium were collected by centrifugation and suspended in 100 µL of 2x SDS-PAGE sample buffer. The cell suspension was vortexed vigorously for 1 min in the presence of 20 µL glass beads, frozen in liquid nitrogen for 5 min, thawed at 37°C for 5 min, and cleared by centrifugation for 10 min. Total soluble protein in 0.5 µL was quantified using the Bio-Rad protein assay reagent. Fifty micrograms of plant or yeast proteins or the indicated volumes of insect cell proteins were separated in 10% (w/v) gels by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl and probed with primary antibody in Tris-buffered saline plus 0.1% Tween 20 (TBST) for 1 h at 22°C. Typically, the primary antibodies to GRIK1 and GRIK2 were used at 1 µg/mL and 0.5 µg/mL, respectively. The antiserum to AL1 was diluted 1:500, and the monoclonal His-tag antibody was diluted 1:2,000. After washing in TBST, the blots were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) diluted 1:2,500 in TBST for 1 h, washed in TBST, and incubated with Pierce's SuperSignal West Pico chemiluminescent substrate. The signals were detected by exposing x-ray films with the membranes.
The central, aboveground part of 5-week-old, soil-grown Arabidopsis rosette seedlings was fixed overnight at 4°C in 4% (w/v) formaldehyde, 15 mM PIPES, pH 6.8, 80 mM KCl, 20 mM NaCl, 0.5 mM EDTA, 2 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 1 mM dithiothreitol, and 0.01 mM sodium acetate. Vertical cross sections were made by embedding the plant material in 5% (w/v) agarose in phosphate-buffered saline (PBS; 20 mM sodium phosphate, pH 7.5, and 130 mM NaCl) and slicing into 50-µm-thick pieces using a Vibratome sectioning system (Technical Products International). Endogenous hydrogen peroxidases were quenched by incubating the sections in 3% (v/v) hydrogen peroxide in methanol for 30 min at 22°C. The sections were then treated with PBS, pH 7.5, containing 1.5% (v/v) normal horse serum and 3% (w/v) bovine serum albumin for 1 h. Primary antibodies at 1 µg/mL in the blocking solution containing 0.1% Tween 20 were incubated with the sections for 1 h. Normal rabbit IgG (Sigma) was used as a negative control. After washing in PBS with 0.1% Tween 20, the sections were incubated in biotinylated horse anti-rabbit IgG antibodies (Vector Laboratories) followed by the Vectastain Elite ABC kit (Vector Laboratories) containing avidin and biotinylated HRP as instructed. The AEC kit (Vector Laboratories) with the substrate 3-amino-9-ethyl carbazole was applied for 10 min, and the sections were then washed in PBS, counter-stained with 1 µg/mL 4',6-diamidino-2-phenylindole for 10 min, and mounted onto a glass slide in 90% (v/v) glycerol in PBS. Immunostained sections were observed with a Nikon Eclipse E800 microscope.
The NucleoSpin RNA Plant kit (CLONTECH) was used to extract RNAs from 100 mg of plant tissue powders. RNA (5 µg) was transcribed to cDNA using PowerScript reverse transcriptase (CLONTECH) and oligo(dT)15 as primer. The cDNAs were diluted to 100 µL with water and 2 µL was subjected to real-time quantitative PCR with gene-specific primers and DyNAmo SYBR Green qPCR reagents (Finnzymes) in Stratagene Mx3000P Real-Time PCR system. All the reactions were performed in triplicate, and mRNA corresponding to the Arabidopsis Act2 gene (At3g18780) was used as reference. Amplification efficiencies for the genes were estimated according to the slopes of their amplification curves (Ramakers et al., 2003
The GRIK1 coding region was PCR amplified from pNSB1185 with the primers 5'-CCGGAATTCATGTTTTGTGATAGT-3' and 5'-CGAAGCTCGAGTCAGCTATGGTTTTG-3'; and the coding region of GRIK2 was obtained from pNSB1184 with the primers 5'-CCGGAATTCATGTTTCGTGATAGT-3' and 5'-CGAAGCTCGAGTTAGTTAGGATCTGA-3'. The DNA fragments were digested with EcoRI and XhoI and fused with the GAL4 DNA binding domain by ligation into the vector pGBKT7 (TRP1; CLONTECH) also digested with EcoRI and SalI. The resulting plasmids, pNSB1398 (GRIK1) and pNSB1399 (GRIK2), were used as baits in yeast strain AH109 (MATa, leu2, trp1, ura3, his3, gal4
The same EcoRI-XhoI-digested DNA fragments were ligated into the yeast expression vector pWS93 (URA3) digested with EcoRI and SalI (Song and Carlson, 1998
In the yeast triple mutant strain MCY5138 (MAT
Proteins with similar amino acid sequences were retrieved from GenBank by performing protein-protein BLAST searches. Sequence alignment of GRIK1, GRIK2, and their plant homologs were done with ClustalX 1.83 and adjusted manually. The kinase domains of the proteins from Arabidopsis, Medicago truncatula, rice (Oryza sativa), Dictyostelium discoideum, zebrafish (Danio rerio), human, Caenorhabditis elegans, Drosophila melanogaster, S. cerevisiae, and Schizosaccharomyces pombe were also aligned and used to build a phylogenetic tree by the neighbor joining method with MEGA 3.1 (Kumar et al., 2004
We thank Drs. David M. Bisaro (The Ohio State University, Columbus, OH), Luisa Lopez-Ochoa, Sharon B. Settlage, Dominique Robertson, and Ralph E. Dewey (North Carolina State University, Raleigh, NC) for their critical comments on this manuscript; Dr. Marian Carlson (Columbia University, New York) for providing the yeast mutant MCY5138 and the plasmid pWS93; David Bisaro for the BCTV strain; Jose T. Ascencio-Ibañez and Luisa Lopez-Ochoa for the protein samples from BCTV-infected Arabidopsis leaves; and Dr. Gerardas Dambrauskas for cloning the full-length GRIK1 and GRIK2 cDNAs and preparing the insect cell expressed GRIK1 and GRIK2 proteins. Received August 21, 2006; accepted October 3, 2006; published October 20, 2006.
1 This work was supported by the National Science Foundation (grant no. IBN–0235251).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Linda Hanley-Bowdoin (linda_hanley-bowdoin{at}ncsu.edu). www.plantphysiol.org/cgi/doi/10.1104/pp.106.088476 * Corresponding author; e-mail linda_hanley-bowdoin{at}ncsu.edu; fax 919–515–2047.
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(Luque et al., 2002
, gal180
