Tomato SlSnRK1 protein interacts with and phosphorylates βC1, a pathogenesis protein encoded by a geminivirus β-satellite.

The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.

In nature, plants are continuously exposed to attacks by a variety of microbial pathogens, including viruses. To combat attack, plants have evolved complex mechanisms to respond to virus challenge, including the hypersensitive response mediated by resistance genes and posttranscriptional gene silencing (Whitham et al., 1994;Vanitharani et al., 2005). The elucidation of hostvirus interactions will be important for providing additional clues to basic compatibility functions as well as host surveillance mechanisms.
Geminiviruses are a group of plant DNA viruses characterized by single-stranded circular genomes encapsidated in twinned icosahedral particles that range in size from 18 to 30 nm (Hanley-Bowdoin et al., 2000;Rojas et al., 2005). They can be divided into four genera (Mastrevirus, Topocuvirus, Curtovirus, and Begomovirus) based on genome structure, insect vectors, and host range (Fauquet and Stanley, 2005). Within the family Geminiviridae, begomoviruses are the most numerous and geographically widespread viruses. Begomoviruses have either monopartite or bipartite genomes (Hanley-Bowdoin et al., 2000;Fauquet et al., 2003). In recent years, a b-satellite molecule, which is a circular single-stranded DNA of approximately 1,350 nucleotides, has been associated with some monopartite begomoviruses, where it is essential for the induction of typical disease symptoms (Saunders et al., 2000(Saunders et al., , 2003Briddon et al., 2001;Jose and Usha, 2003;Cui et al., 2004). Full-length b-satellite molecules encode an approximately 13.5-kD protein known as bC1 on the complementary sense strand. bC1 is a pathogenicity determinant and a suppressor of RNA silencing (Jose and Usha, 2003;Cui et al., 2004Cui et al., , 2005Saunders et al., 2004;Qian and Zhou, 2005;Saeed et al., 2005;Gopal et al., 2007;Kon et al., 2007). Previous studies showed that bC1 interacts with ASYMMETRIC LEAVES1 to alter leaf development and suppress selected jasmonic acid responses (Yang et al., 2008). bC1 also interacts with a host ubiquitinconjugating enzyme, SlUBC3, and modifies the host ubiquitination system (Eini et al., 2009). Here, we show that bC1 of the tomato yellow leaf curl China b-satellite (TYLCCNB) interacts with a tomato (Solanum lycopersicum) SUCROSE-NONFERMENTING1 (SNF1)-related kinase (SlSnRK1). We also demonstrate that SlSnRK1 phosphorylates the bC1 protein in vitro and that mutations in the primary phospho residues impact the pathogenicity function of bC1.

bC1 Interacts with Tomato SlSnRK1
To identify host proteins that interact with the bC1 protein of TYLCCNB (TYLCCNB-bC1), we performed a yeast two-hybrid screen of a tomato cDNA library fused to the GAL4 activation domain using TYLCCNB-bC1 fused to the GAL4 DNA-binding domain as bait. From a total of 5 3 10 5 independent, double transformants assayed for His prototrophy and a-galactosidase activity, one cDNA clone that interacts with bC1 was identified. The clone displayed 100% sequence identity with a cDNA encoding a tomato SNF1-related kinase (AF143743; Bradford et al., 2003), which we have named SlSnRK1. Based on the complete nucleotide sequence of AF143743, the full-length coding sequence of SlSnRK1 was amplified from tomato cDNA using primers SlSnRK1-1F-Ec and SlSnRK1-1R-Ba, and its interaction with bC1 was confirmed by the yeast twohybrid system (Fig. 1A).
The full-length SlSnRK1 cDNA is 1,545 nucleotides and encodes a protein of 514 amino acids (58,824 D). It is closely related to SnRK1 in Nicotiana benthamiana, NPK5 in tobacco (Nicotiana tabacum), and AKIN11 in Arabidopsis (Arabidopsis thaliana), with identities of 95%, 87%, and 78%, respectively ( Fig. 1B; Supplemental Fig. S1). As a typical member of the SnRK1 subfamily, SlSnRK1 encodes the a-subunit of the SnRK1 heterotrimer, which is a key regulator of the plant response to starvation and metabolic stress (Bradford et al., 2003;Halford and Hey, 2009). SlSnRK1 contains a conserved kinase domain (KD) in the N terminus, an internal ubiquitin-associated domain (UBA), and an autoinhibitory sequence (AIS) domain, as well as a C-terminal domain (CTD) that is responsible for b-subunit binding and formation of the SnRK1 complex (Fig. 1C).
bC1 and SlSnRK1 Interaction in Planta Bimolecular fluorescence complementation (BiFC) was performed in agro-infiltrated N. benthamiana leaves to test for interaction between bC1 and SlSnRK1 in plant cells. For this assay, bC1 was fused to the N-terminal fragment of yellow fluorescent protein (YFPN; pbC1-YFP N ) and SlSnRK1 was fused to the C-terminal fragment of YFP (YFPC; pSlSnRK1-YFP C ). Pair-wise expression of pbC1-YFP N and pSlSnRK1-YFP C resulted in a YFP fluorescence signal in the cytoplasm of agroinfiltrated cells at 72 h post infiltration, but no YFP fluorescence was observed when pbC1-YFP N and pYFP C , or pSlSnRK1-YFP C and pYFP N , were coexpressed (Fig. 2). These results con-firm that the bC1 protein interacts with SlSnRK1 in plant cells.

Subcellular Localization and Expression Pattern of SlSnRK1
Subcellular localization of GFP-tagged SlSnRK1 was examined by agroinfiltration of N. benthamiana epidermal cells (Fig. 3). Green fluorescence was detected in the cytoplasm and nuclei of cells expressing GFP-SlSnRK1. A similar fluorescence pattern was observed in cells expressing the GFP control protein. These data are consistent with the localization of GFP-tagged SlSnRK1 to both the cytoplasmic and nuclear compartments of plant cells.
To determine the expression pattern of SlSnRK1 mRNA, quantitative reverse transcription (RT)-PCR was performed using total RNA from various tomato tissues as template. The highest level of SlSnRK1 mRNA was detected in the flower, with intermediate levels in the leaf and the lowest levels in the root and stem (Fig. 4A). We also compared SlSnRK1 transcript Figure 1. A, Interaction between SlSnRK1 and TYLCCNB-bC1 in a yeast two-hybrid system. Yeast strain AH109 cotransformed with the indicated plasmids was spotted with 10-fold serial dilutions on synthetic dextrose (SD)/2His/2Leu/2Trp medium containing 5 mM 3-aminotriazole (3-AT). B, Phylogenetic tree based on SlSnRK1 amino acid sequences shown in Supplemental Figure S1 using Clustal analysis with PAM250 residue weight (DNASTAR). C, Schematic representation of SlSnRK1. Putative functional domains are indicated. The KD, UBA, AIS, and CTD responsible for b-subunit binding and formation of the SnRK1 complex were deduced from InterProScan online software (http://www.ebi.ac.uk/Tools/InterProScan/) and further confirmed by comparison with previous descriptions (Crute et al., 1998;Hardie, 2007). [See online article for color version of this figure.] levels in tomato leaves inoculated with TYLCCNV/ TYLCCNB or with TYLCCNV alone by quantitative RT-PCR. Higher levels of SlSnRK1 mRNA were also detected in TYLCCNV/TYLCCNB-infected than in TYLCCNV-infected leaves at 3 d post inoculation (DPI; Fig. 4B). These results suggested that TYLCCNB stimulates the accumulation of the SlSnRK1 mRNA.

Identification of the Domains Necessary for bC1 and SlSnRK1 Interaction
To locate the domains necessary for bC1 and SlSnRK1 interaction, we tested eight deletion mutants for bC1 and nine deletion mutants for SlSnRK1, respectively ( Fig. 5) in yeast two-hybrid assays. As shown in Figure 5A, yeast transformants harboring SlSnRK1 mutants M1 (amino acids 281-514), M2 (amino acids 1-339), M4 (amino acids 1-460), M5 (amino acids 1-360 fused with 450-514), or M6 (amino acids 1-290 fused with 339-514) grew on TDO/Aba+ (for synthetic dextrose/2His/2Leu/2Trp medium in the presence of 90 or 120 ng mL 21 aureobasidin A) plates, indicative of interaction with bC1. In contrast, mutant M3 (amino acids 1-280) failed to interact with bC1. No bC1-binding activity was detected for the kinase domain alone (residues 1-280), and deletion of the kinase domain did not affect the interaction be-tween SlSnRK1 and bC1. Based on these results, we concluded that the kinase domain is not involved in bC1 binding. Instead, our data suggested that the central and C-terminal regions are involved in binding bC1.
To further define the bC1-binding domain, we constructed three mutants (M7-M9) that divided SlSnRK1-M1 into known functional domains. Yeast two-hybrid assay revealed that both M7 (residues 281-339, containing the UBA domain) and M8 (residues 340-449, containing the AIS domain) retained significant bC1-binding activity, while M9 (residues 450-514, containing the CTD) did not show any binding activity (Fig. 5A). These results suggested that the UBA and AIS domains in SlSnRK1 can each interact with bC1.
All of the bC1 mutants including deletions of only 10 amino acids of either the N or C terminus failed to interact with full-length SlSnRK1 (Fig. 5B), indicating that full-length bC1 is required for interaction with SlSnRK1.

Plant SnRK1 Affects TYLCCNV/TYLCCNB Infection and Alters Viral DNA Accumulation
To assess the biological significance of SlSnRK1-bC1 interactions in vivo, we inoculated wild-type and transgenic N. benthamiana plants carrying an Arabidopsis antisense SnRK1 expression cassette (AS-12) or an Arabidopsis sense SnRK1 expression cassette (S-5; Hao et al., 2003) with TYLCCNV/TYLCCNB and monitored infection over time (Carvalho et al., 2008b). Changes in SnRK1 expression altered the timing of symptom appearance, with overexpressing plants developing symptoms later and silenced plants showing symptoms earlier than wild-type plants (Fig. 6A). The shift in the timing of symptom development was readily apparent when the infectivity data were expressed as days post inoculation to reach 50% of symptomatic plants (DPI 50%), with values of 7.7, 5.8, and 5.1 DPI 50% for SnRK1-overexpressing, silenced, and wild-type plants, respectively (Fig. 6B). However, both wild-type and transgenic plants displayed similar symptoms during the late stage of infection. DNA gel-blot analysis showed that SnRK1overexpressing plants accumulated less viral DNA and that SnRK1-silenced plants accumulated more viral DNA when compared with wild-type plants infected with TYLCCNV/TYLCCNB (Fig. 6C). Together, these results suggested that increasing SnRK1 levels reduce infection efficiency while lowering SnRK1 levels enhances efficiency. To rule out the possible functional differences between Arabidopsis SnRK1 (AKIN11) and SlSnRK1, we showed that bC1 also interacts with AKIN11 (Supplemental Fig. S2).

Impacts of SlSnRK1 Kinase Activity by bC1
We next asked if bC1 can impact SlSnRK1 kinase activity in yeast cells.
In initial experiments, we tested whether SlSnRK1 can complement the yeast snf1 deletion strain Dsnf1 BY4741, which cannot grow on medium containing a carbon source other than Glc. Expression of SlSnRK1 in Dsnf1 BY4741 restored growth on synthetic complete medium containing 2% (w/v) Gal and 2% (w/v) Suc as carbon sources, but Dsnf1 BY4741 transformed with the empty expression plasmid pESC-Ura or a plasmid expressing the kinase-dead mutant, CTU-SlSnRK1 K48R , failed to grow on the medium (Fig. 7A). Thus, SlSnRK1 can functionally complement SNF1 in yeast and SlSnRK1 kinase activity is essential for complementation. We found that Dsnf1 BY4741 cotransformed with SCU-SlSnRK1 and SCL-bC1 grew similarly as Dsnf1 BY4741 cotransformed SCU-SlSnRK1 and pESC-Leu (positive control) on medium containing Gal and Suc as carbon sources, but Dsnf1 BY4741 cotransformed with pESC-Ura and SCL-bC1 (negative control) could not grow on the medium (Fig. 7B), suggesting that bC1 cannot inhibit SlSnRK1 activity.

SlSnRK1 Phosphorylates bC1 Mainly on Thr at Position 78 and Ser at Position 33 in Vitro
To test if SlSnRK1 phosphorylates bC1 protein, both proteins were expressed in Escherichia coli as glutathione S-transferase (GST) fusions. The SAMS peptide, HMRSAMSGLHLVKRR, which is a specific and sensitive substrate for the SNF1/AMPK/SnRK1 kinases (Sugden et al., 1999), was also fused to GST and used as a positive control, while GST alone was used as a negative control. The Arabidopsis GRIK1 protein has been reported to act as an upstream activating kinase of SnRK1 by phosphorylating its activation loop (Shen et al., 2009), so we used GST-tagged GRIK1 protein to activate SlSnRK1 during the in vitro kinase reactions. Because the full-length SlSnRK1 protein was insoluble, a truncated SlSnRK1 containing the KD and UBA domain (SlSnRK1-KD) was used for the in vitro phosphorylation assays. Recombinant bC1, SAMS, and GST proteins were separately incubated with GRIK1 and SlSnRK1-KD proteins under the same reaction conditions. Phosphorylation of bC1 was clearly ob- served after coincubation with SlSnRK1-KD, whereas GST was not phosphorylated (Fig. 8A), indicating that bC1 is a SlSnRK1-KD substrate in vitro. To rule out the possibility that phosphorylation of bC1 was catalyzed by GRIK1, an additional reaction containing GRIK1 and bC1 in the absence of SlSnRK1-KD was carried out. Autoradiography only detected a radiolabeled band for GRIK1, indicating that the viral protein bC1 is specifically phosphorylated by SlSnRK1.
To determine the bC1 residue(s) phosphorylated by SlSnRK1 in vitro, we first analyzed the coding sequence of full-length bC1 using NetPhos 2.0 for potential phosphorylation sites (http://www.cbs.dtu. dk/services/NetPhos/). The analysis revealed that the Thr at position 78 (Thr-78) and the Ser at position 33 (Ser-33) are potential phosphorylation sites for Ser/Thr kinases (data not shown). We generated six bC1 point mutants, bC1 S33A , bC1 S33D , bC1 T78A , bC1 T78D , bC1 S33A/T78A , and bC1 S33D/T78D , in which Ser-33 or Thr-78 of bC1 was individually or simultaneously replaced by Ala to eliminate phosphorylation Relative mRNA levels in tomato tissues were normalized using EF-1a mRNA as a reference. Values are means of three independent experiments. Different lowercase letters above the bars denote significant differences (Fisher's LSD method; P , 0.05). or by Asp to mimic constitutive phosphorylation (Waigmann et al., 2000;Karger et al., 2003;Trutnyeva et al., 2005). The mutant proteins were expressed in E. coli as GST fusion proteins and used in kinase assays. As shown in Figure 8, B and C, mutations S33A, S33D, T78A, T78D, S33A/T78A, and S33D/T78D significantly reduced the level of bC1 phosphorylation relative to the wild-type protein. These differences were not due to different loading amounts of bC1 and its mutants (Fig. 8B). The reductions in radioactive signals for bC1 S33A and bC1 S33D were stronger than for bC1 T78A and bC1 T78D . These results indicated that both bC1 Ser-33 and Thr-78 are SlSnRK1 phosphorylation sites. Replacement of Ser-33 or Thr-78 with Asp did not promote bC1 phosphorylation of the other site, indicating that the two sites are unlikely to be synergistic for phosphorylation. Both double mutants bC1 S33A/T78A and bC1 S33D/T78D retained low but measurable phosphorylation signals (37%-28% of the wild-type bC1; Fig. 8C), suggesting that other bC1 residues might also be phosphorylated by SlSnRK1 in vitro.

Effect of bC1 Phosphorylation Site Mutations on Virus Infection and Viral DNA Accumulation
To assess the role of bC1 phosphorylation by SlSnRK1, Ser-33 and/or Thr-78 mutations were introduced into the bC1 gene of a satellite replicon. Six mutant constructs were generated, including single replacements at Ser-33 or Thr-78 to Ala (S33A and T78A) or Asp (S33D and T78D) as well as double mutants (S33A/T78A and S33D/T78D). Infectious clones of these mutants and wild-type TYLCCNB were used to inoculate N. benthamiana plants together with TYLCCNV. Delay of virus infection associated with mild symptoms was observed in plants inoculated with the Asp mutants TYLCCNB-S33D, TYLCCNB-T78D, and TYLCCNB-S33D/T78D (Fig. 9, A and B). When the infectivity data were expressed as DPI 50%, Interaction between SlSnRK1 and bC1 the bC1 phosphomimic mutations (S33D, T78D, and S33D/T78D) reduced the efficiency of virus infection (Fig. 9C). DNA gel-blot analysis showed that viral DNA accumulation was lower in plants coinoculated with TYLCCNV and a bC1 phosphomimic mutant (S33D, T78D, or S33D/T78D) than in plants coinoculated with TYLCCNV and wild-type TYLCCNB (Fig.  9D). In contrast, symptom appearance and severity associated with the TYLCCNB Ala mutants (S33A, T78A, S33A/T78A) resembled wild-type TYLCCNB (Fig. 9, A and B), but viral DNA levels were higher in plants infected by the Ala mutants versus the wildtype satellite (Fig. 9D). These data indicated that mutations in the primary residues phosphorylated by SnRK1 in vitro impact the pathogenicity function of bC1.

DISCUSSION
Geminiviruses infect a broad variety of plants and induce a wide range of symptoms (Hanley-Bowdoin et al., 2000). Recent studies indicated that they can evade the plant immune system by interfering with host antiviral pathways Wang et al., 2003;Florentino et al., 2006;Piroux et al., 2007). In response, the plant hosts have evolved diverse innate defense mechanisms to counter these challenges (Voinnet, 2001;Xie and Guo, 2006). Many host factors have been shown to be hijacked or coopted by geminiviruses to facilitate infection (Kong et al., 2000;Egelkrout et al., 2001;Carvalho et al., 2008a). In contrast, few host factors have been shown to participate in plant antiviral processes. For example, interaction between the RepA protein of Wheat dwarf virus (WDV) and a wheat NAC domain protein (GRAB) severely impairs WDV replication in cultured wheat (Triticum aestivum) cells (Xie et al., 1999). Similarly, the expression of sense RNAs of tomato SUMO (LeSUMO) impairs tomato golden mosaic virus (TGMV) replication, suggesting that plant SUMO may also play an important role in the plant antiviral defense response (Castillo et al., 2004).
TYLCCNV is a monopartite begomovirus associated with a b-satellite (TYLCCNB) identified in China (Zhou et al., 2003). We demonstrated previously that TYLCCNV alone produces asymptomatic infections in tobacco, tomato, and petunia (Petunia hybrida) and that TYLCCNB is required for the production of leaf curl symptoms in these hosts (Cui et al., 2004). These studies showed that the bC1 protein encoded by TYLCCNB is a pathogenicity factor that is necessary for symptom production. In this study, we showed that TYLCCNB-bC1 interacts with a tomato SNF1related kinase designated as SlSnRK1 by yeast twohybrid analysis and BiFC assay.
Yeast SNF1, mammalian AMPK, and plant SnRK1 are a group of Ser/Thr protein kinases that are conserved in all eukaryotes and have similar subunit compositions, subunit structures, and common kinase cascades (Hardie et al., 1998;Halford et al., 2003Halford et al., , 2004Hardie, 2007;Polge and Thomas, 2007;Baena-González and Sheen, 2008). In plants, SnRK1 is widely recognized to be involved in various physiological processes, including nutrient and energy sensing, global regulation of metabolism, control of the cell cycle, modulation of development, and response to abiotic or biotic stress (Baena-González et al., 2007). Although a number of SnRK1 functions have been characterized, information about SnRK1 function against pathogen infection is limited. Hao et al. (2003) showed that the AL2 protein from TGMV (genus Begomovirus) and the L2 protein from Beet curly top virus (genus Curtovirus) interact with Arabidopsis SnRK1 (AKIN11) and that AL2 and L2 inactivate SnRK1, leading to enhanced susceptibility. These results suggested that metabolic alterations mediated by SnRK1 may contribute to plant innate antiviral defenses and that SnRK1 inactivation by AL2 and L2 is a counterdefense measure. Although bC1 also binds to SnRK1, we found that bC1 does not inhibit SnRK1 kinase activity and, instead, is phosphorylated by SlSnRK1. The bC1 phosphomimic mutants S33D and T78D attenuate symptoms, delay viral infection, Figure 7. SlSnRK1 functionally complements SNF1 in yeast (A), and TYLCCNB-bC1 does not inhibit SlSnRK1 activity in yeast (B). Cells of Dsnf1 BY4741 transformed with the indicated plasmids were spotted with serial 10-fold dilutions on selective synthetic complete medium. SlSnRK1 was expressed from a high-copy plasmid (SCU-SlSnRK1) or a low-copy plasmid (CTU-SlSnRK1). bC1 was expressed from a highcopy plasmid (SCL-bC1), and SlSnRK1 K48R was expressed from a lowcopy plasmid (CTU-SlSnRK1 K48R ). Dsnf1 BY4741 transformed with the empty vector pESC-Ura or cotransformed with pESC-Ura and SCL-bC1 was used as a negative control. Dsnf1 and reduce viral DNA accumulation, while the Ala mutants S33A and T78A enhance viral DNA accumulation, suggesting that phosphorylation of the bC1 protein negatively impacts its function as a pathogenicity determinant.
Protein phosphorylation may be a common process in response to virus challenge by plants. Our data support a model in which phosphorylation of bC1 by SlSnRK1 is a counterdefense response against virus infection by the host. We demonstrated previously that transgenic N. benthamiana, tobacco, and Arabidopsis plants expressing the TYLCCNB bC1 gene are stunted and show leaf cupping and curling. The resulting "symptoms" are much more severe than those associated with TYLCCNV plus TYLCCNB infection, demonstrating that bC1 is very toxic to plants (Cui et al., 2004;Yang et al., 2008). SnRK1 phosphorylation of bC1 may be used by plants to overcome its detrimental effects. This idea is supported by our observation that reducing SnRK1 expression enhances the efficiency of TYLCCNV plus TYLCCNB infection and increases viral DNA accumulation.
An unanswered question is how the phosphorylation of bC1 impacts its pathogenicity function. We showed previously that the bC1 protein binds to DNA in a sequence-nonspecific manner, functions as a suppressor of RNA silencing, and is a pathogenicity protein that plays a vital role in symptom induction by suppression of the silencing defenses in plants (Cui et al., 2005). Phosphorylation of proteins can regulate their nucleic acid-binding properties (Boyle et al., 1991;Mayrand et al., 1993) and interactions between viral RNA and replication proteins of positive-strand RNA viruses (Shapka et al., 2005;Stork et al., 2005). Phosphorylation of bC1 may inhibit its ability to bind nucleic acid, which may negatively impact bC1 function as an RNA-silencing suppressor and result in attenuating viral infection. Alternatively, the stability of bC1 protein could be influenced by its phosphorylation status. Previous studies demonstrated that phosphorylation of hepatitis C virus NS5A (Pietschmann et al., 2001), turnip yellow mosaic virus 66K protein (Héricourt et al., 2000;Jakubiec et al., 2006), or tobamovirus MP (Kawakami et al., 1999) can affect viral protein and Thr-78. The asterisk represents GST contaminants during purification, and no phosphorylation signal was detected on them. C, The radioactive signals shown in B were quantified by ImageQuant TL V2003 software. All data represent means 6 SE of three replicate experiments. Different lowercase letters above the bars denote significant differences (Fisher's LSD method; P , 0.05).
stability. In addition, protein phosphorylation often plays a role in ubiquitin-mediated proteolysis, and SCF (one type of multisubunit ubiquitin-protein ligase [E3]) degradation pathways are mediated by phosphorylation-dependent substrate recognition (Kong and Chock, 1992;Clurman et al., 1996;Won and Reed, 1996;Musti et al., 1997;Willems et al., 1999;Pickart, 2001;Feng et al., 2004;Gao et al., 2004;Dreher and Callis, 2007). The recent discovery that bC1 protein is degraded by the 26S proteasome (Yang et al., 2008) indicates that SlSnRK1 may inter-act with and phosphorylate bC1 for degradation by the 26S proteasome, leading to the attenuation of symptoms and reduction of the efficiency of viral infection.
In conclusion, we have demonstrated that tomato SlSnRK1 protein interacts with and phosphorylates bC1, a pathogenicity factor encoded by a geminivirus b-satellite. Future studies will determine whether the phosphorylation of bC1 negatively impacts its function as a RNA-silencing suppressor and/or mediates its degradation by the 26S proteasome.  TYCCNV and TYCCNB or its mutants  (TYLCCNB-S33A, TYLCCNB-S33D, TYLCCNB-T78A, TYLCCNB-T78D, TYLCCNB-S33A/T78A, and TYLCCNB-S33D/T78D) at 17 DPI. CK2 indicates the mock-inoculated plant. B, Infection course of TYCCNV and TYCCNB or its mutants. Values represent percentages of systemically infected plants at different DPI and are given as means 6 SD of triplicate experiments. In each experiment, 12 plants were inoculated. C, DPI 50% after inoculation of TYLCCNV and TYLCCNB or its mutants. The data are means 6 SD of triplicate experiments. Different lowercase letters above the bars denote significant differences (Fisher's LSD method; P , 0.05). D, TYLCCNV and TYLCCNB DNA levels in wild-type and transgenic plants expressing antisense SnRK1 (AS-12) or sense SnRK1 (S-5) at 8 and 15 DPI. After infection, total DNA from a whole-plant mixture was used for DNA gel blotting. Blots were probed with the CP gene sequence of TYLCCNV (top) or the full-length sequence of TYLCCNB (middle). An ethidium bromide-stained gel shown below the blots provides a DNA-loading control. The positions of single-stranded (ssDNA) and subgenomic (sgDNA) forms of TYLCCNV and TYLCCNB are indicated.

Plant Material, Growth Conditions, and Genotyping
Tomato (Solanum lycopersicum 'Hongbaoshi') was used to construct the tomato cDNA library. Antisense (AS-12) and sense (S-5) AKIN11 transgenic Nicotiana benthamiana lines  were kindly provided by Dr. David M. Bisaro. Plants were grown in 10-cm pots filled with a mixture of 60% vermiculite and 40% meadow soil in a growth chamber at 25°C under longday conditions (16 h of light/8 h of dark).

Plasmid Construction
The plasmids used in this study are listed in Supplemental Table S1. The primers used for mutagenesis and subcloning are given in Supplemental Table S2.
The coding sequences of the full-length Arabidopsis (Arabidopsis thaliana) AKIN11 were amplified separately using the primer pairs listed in Supplemental Table S2. The AKIN11 PCR fragments were digested with EcoRI/BamHI and cloned into the vector pGBKT7 and vector pGADT7 (Clontech) to generate the recombinant plasmids listed in Supplemental Table S1.
For the production of BiFC vectors, the full-length coding sequence of bC1 was amplified using the primers listed in Supplemental Table S2 and cloned into the PacI-AscI site of p2YN (Yang et al., 2007) as a fusion with the N-terminal fragment of YFP, resulting in pbC1-YFP N . The full-length coding sequence of tomato SlSnRK1 was amplified using the primers listed in Supplemental Table S2 and cloned into the PacI-AscI site of p2YC (Yang et al., 2007) as a fusion with the C-terminal fragment of YFP, resulting in pSlSnRK1-YFP C .
For subcellular localization studies, SlSnRK1 was tagged at its N terminus with GFP by inserting PCR-amplified SlSnRK1 cDNAs using the primer pair listed in Supplemental Table S2 into the BamHI-PstI site of pCHF3-GFP to produce GFP-SlSnRK1.
For yeast complementation studies, the full-length coding sequence of tomato SlSnRK1 was amplified using the primer pair listed in Supplemental  Table S2 and cloned into the EcoRI-SpeI site of pESC-Ura (2m origin, URA3 selection marker; Stratagene) or the KpnI-BamHI site of pYC2/CT-Ura (CEN6/ ARSH4 origin, URA3 selection marker; Invitrogen), resulting in SCU-SlSnRK1 or CTU-SlSnRK1, respectively. The dead kinase mutant SlSnRK1 K48R was generated by overlapping PCR (Tao et al., 2002) using the complementary primer pairs listed in Supplemental Table S2. The PCR fragments were digested with KpnI-BamHI and cloned into the vector pYC2/CT-Ura (Invitrogen) to generate recombinant plasmid CTU-SlSnRK1 K48R . The coding sequences of intact bC1 were amplified using the primers listed in Supplemental Table S2 and cloned into the BamHI-SalI site of pESC-Leu (2m origin, LEU2 selection marker; Stratagene), resulting in SCL-bC1.
The plasmid pNSB1554 harboring GST-fused GRIK1 was constructed previously (Shen et al., 2009). The plasmid pGEX-KG-SAMS expressing the positive control peptide SAMS for kinase assay was kindly provided by Dr. David M. Bisaro.

Yeast Two-Hybrid Screens
The construction and screening of the tomato cDNA library and the analyses of positive interactions were performed according to the BD Matchmaker Library Construction and Screening Kits User Manual (Clontech). Total RNAs were extracted from tomato seedlings using TRIzol (Invitrogen), and mRNA (1.0 mg) was isolated with an mRNA isolation kit (Promega) and used for cDNA library construction. The tomato cDNA library was screened with BD-bC1 as bait in Saccharomyces cerevisiae strain AH109 (Clontech), and positive clones were selected on a His-deficient medium, confirmed by b-GAL assays.
The plasmids BD-bC1 and AD-SlSnRK1 were cotransformed into S. cerevisiae strain AH109. Plasmids BD-53 and AD-T served a positive controls, and plasmids BD-Lam and AD-T, BD-bC1 and AD, and BD and AD-SlSnRK1 were used as negative controls. Transformants were grown at 30°C for 72 h on synthetic medium lacking Leu and Trp and then transferred to the medium lacking His, Leu, and Trp and containing 5 mM 3-aminotriazole to identify binding activity. Three independent experiments were performed to confirm the result.
The recombinant plasmids AD-AKIN11 and BD-bC1, SlSnRK1 deletion mutant and AD-bC1, and bC1 deletion mutant and BD-SlSnRK1 were cotransformed into S. cerevisiae Y2HGold cells (Clontech). Transformants were transferred to TDO/AbA to identify binding activity.
BiFC Assay pbC1-YFP N and pSlSnRK1-YFP C were introduced individually into Agrobacterium tumefaciens strain C58C1 by electroporation. BiFC experiments were performed as described previously (Yang et al., 2007). YFP fluorescence was observed and photographed by confocal microscopy (Leica TCS SP5) at 48 to72 h after infiltration.

Subcellular Localization of Proteins
pCHF3-GFP and pCHF3-GFP-SlSnRK1 were introduced individually into A. tumefaciens strain EHA105 by electroporation. Leaves of 4-week-old N. benthamiana plants were infiltrated with the A. tumefaciens harboring the constructs as described (Liu et al., 2009). About 48 h after infiltration, 1-cm 2 leaf explants were excised and GFP fluorescence was examined in epidermal cells by confocal microscopy (Leica TCS SP5).

Real-Time RT-PCR Analyses
Total RNA was extracted using TRIzol (Invitrogen). The first-strand cDNA was synthesized as described (Burton et al., 2000;Liu et al., 2002;Tao and Zhou, 2004). Real-time RT-PCR were performed as described (Huang et al., 2009) using primer pair SlSnRK1-rt-ORF-1F/SlSnRK1-rt-UTR-1r specific for SlSnRK1. The primer SlSnRK1-rt-UTR-1r annealed to the untranslated region of SlSnRK1 to ensure that only the SlSnRK1 mRNA gene was amplified. The EF-1a gene was used as an internal control.

Protein Expression and Kinase Assay
Recombinant proteins were produced in Escherichia coli strain BL21 (DE3) induced with 0.5 mM isopropyl b-D-thiogalactoside for 16 h at 16°C. Bacterial cells were collected and disrupted by sonication. The GST-fused proteins were purified using GST-binding resin (Novagen, Merck) according to the manufacturer's instructions.
In vitro kinase assays were performed as described (Lin et al., 2009) with minor modifications. Purified proteins including GST-GRIK and GST-SlSnRK1-KD were coincubated with GST-tagged wild-type bC1 or its mutants (bC1 S33A , bC1 S33D , bC1 T78A , bC1 T78D , bC1 S33A/T78A , and bC1 S33D/T78D ) in reaction buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 10 mM ATP, 0.1 mM CaCl 2 , and 2 mM dithiothreitol) in a total volume of 20 mL. GST-SAMS and GST were used as positive and negative controls, respectively. Reactions were initiated by the addition of 5 mCi of [g-32 P]ATP and transferred to 30°C for 30 min. Loading buffer (63 SDS; 4 mL) was added to terminate the reactions. After boiling at 95°C for 5 min, proteins were separated on a 12% SDS-PAGE gel followed by staining with Coomassie Brilliant Blue R-250. Radioactive signals were visualized through autoradiography and quantified by ImageQuant TL V2003 software (GE Healthcare).

Virus Inoculation and Infectivity Tests
The infectious clones of TYLCCNB mutants were introduced into A. tumefaciens strain EHA105 by the electroporation method. Wild-type or transgenic N. benthamiana plants expressing Arabidopsis antisense SnRK1 (AS-12) or Arabidopsis sense SnRK1 (S-5) were agroinoculated with an overnight culture of A. tumefaciens carrying TYLCCNV and TYLCCNB (pBinPLUS-1.7A and pBinPLUS-2b; Cui et al., 2004) or TYLCCNB mutants. The course of infection was monitored as described (Carvalho et al., 2008b). DPI 50% was determined using data from three independent experiments.

DNA Gel Blotting
Total DNA was extracted from leaves of tobacco (Nicotiana benthamiana) plants as described previously (Zhou et al., 2001). DNA gel blotting was performed as described previously (Cui et al., 2004). Genomic DNA was stained using ethidium bromide as a loading control.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AF143743 (SlSnRK1).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. SlSnRK1 has sequence homology with other SnRK1 a-subunits and AMPK from Caenorhabditis elegans.
Supplemental Figure S2. Interaction between AKIN11 and bC1 in yeast.
Supplemental Table S1. Recombinant plasmids used in this study.
Supplemental Table S2. Primers used in plasmid construction and other experiments.