The β -subunit of the SnRK1 complex is phosphorylated by the plant cell death suppressor

The protein kinase Adi3 is a known suppressor of cell death and loss of its function has been correlated with cell death induction during the tomato ( Solanum lycopersicum ) resistance response to its pathogen Pseudomonas syringae pv. tomato . However, Adi3 downstream interactors that may play a role in cell death regulation have not been identified. We used a yeast two-hybrid screen to identify the plant SnRK1 (Sucrose non-Fermenting-1-Related Protein Kinase 1) protein as an Adi3 interacting protein. SnRK1 functions as a regulator of carbon metabolism and responses to biotic and abiotic stresses. SnRK1 exists in a heterotrimeric complex with a catalytic α -subunit (SnRK1), a substrate interacting β -subunit, and a regulatory γ -subunit. Here we show that Adi3 interacts with, but does not phosphorylate the SnRK1 α subunit. The ability of Aid3 to phosphorylate the four identified tomato β -subunits was also examined and it was found that only the Gal83 β -subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as Ser26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates it contains multiple phosphorylation sites, one of which is Ser26. An active SnRK1 complex containing Gal83 as the β -subunit and Snf4 as the γ -subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1, and suggests this function may be related to the cell death suppression activity of Adi3.

complex with a catalytic α -subunit (SnRK1), a substrate interacting β -subunit, and a regulatory γ -subunit. Here we show that Adi3 interacts with, but does not phosphorylate the SnRK1 α subunit. The ability of Aid3 to phosphorylate the four identified tomato β -subunits was also examined and it was found that only the Gal83 β -subunit was phosphorylated by Adi3. This phosphorylation site on Gal83 was identified as Ser26 using a mutational approach and mass spectrometry. In vivo expression of Gal83 indicates it contains multiple phosphorylation sites, one of which is Ser26. An active SnRK1 complex containing Gal83 as the β -subunit and Snf4 as the γ -subunit was constructed to examine functional aspects of the Adi3 interaction with SnRK1 and Gal83. These assays revealed that Adi3 is capable of suppressing the kinase activity of the SnRK1 complex through Gal83 phosphorylation plus the interaction with SnRK1, and suggests this function may be related to the cell death suppression activity of Adi3. al., 2012). Thus, SnRK1 appears to be a key regulator connecting metabolism and stress responses in plants (Halford and Hey, 2009;Hey et al., 2010).
The SnRK1 (and Snf1/AMPK) complex exists as a heterotrimer of an α -subunit Ser/Thr kinase called SnRK1, one of several possible β -subunits (Sip1, Sip2, or Gal83 in yeast), and a γ subunit called Snf4 (Halford and Hey, 2009;Coello et al., 2010). The γ -subunit has been shown to regulate kinase activity of the complex (Jiang and Carlson, 1996), while the β -subunits regulate complex substrate specificity and cellular localization (Mitchelhill et al., 1997;Vincent and Carlson, 1999;Vincent et al., 2001Vincent et al., , 2001Warden et al., 2001). The signaling mechanisms exerted on the β -subunits for controlling function are not fully understood. But, at least for yeast Sip1 and the human β -subunit AMPKβ1, phosphorylation appears to be involved in controlling β -subunit function (Warden et al., 2001;Hedbacker et al., 2004). Recently, two kinases have been shown to phosphorylate yeast Gal83, but a connection to function has not been shown (Mangat et al., 2010) and it has yet to be shown that a plant β -subunit is phosphorylated. Here we present data showing that Adi3 interacts with the tomato SnRK1 α -subunit and the Gal83 β subunit, that Adi3 can only phosphorylate Gal83, and we show that Adi3 can inhibit the kinase activity of the SnRK1 complex.

Identification of SnRK1 as an Adi3 Interacting Protein.
In an effort to identify Adi3-interacting proteins we carried out a yeast two-hybrid (Y2H) screen using a cDNA prey library that has been previously used to identify proteins that interact with the tomato resistance protein kinase Pto (Zhou et al., 1995). Approximately 15 million yeast transformants were screened for Adi3-interacting proteins using selection on Leu-plates and 1,366 transformants were followed-up in a LacZ screen. The prey inserts from 85 random positive clones were sequenced and screened against GenBank by BLAST for identification. Of these clones, SnRK, encoding the α -subunit of the SnRK1 protein complex, was identified four times. The SnRK insert in the prey library was a partial ORF and a full-length ORF was identified by searching the tomato EST data base (http://solgenomics.net/) by BLAST with the ORF and this sequence was amplified from tomato leaf tissue RNA by RT-PCR. A BLAST search against GenBank with the full-length SnRK sequence showed that it was identical to a previously identified tomato SnRK cDNA (Bradford et al., 2003). In Arabidopsis, SnRK proteins are separated into three distinct families, SnRK1, SnRK2, and SnRK3 (Halford and Hey, 2009). BLAST and alignment comparison of the protein encoded by the SnRK sequence cloned here with members of the Arabidopsis SnRK (AtSnRK) family indicated that it belongs to the SnRK1 family (Supplemental Fig. S1). The tomato gene identified here will be referred to as SlSnRK1 throughout this study.
The full-length SlSnRK1 ORF was used to confirm the Y2H interaction with Adi3 and test the interaction with kinase activity mutants of Adi3. SlSnRK1 does not autoactivate in the Y2H assay when expressed from either the prey or bait vectors (Fig. 1A). Our previous studies have shown that mutation of the Pdk1 phosphorylation site on Adi3 (S539) to Asp (Adi3 S539D ) confers constitutive kinase activity on Adi3, and mutation of Lys337 to Gln (Adi3 K337Q ) in the ATPbinding pocket eliminates Adi3 kinase activity (Devarenne et al., 2006). The interaction of SlSnRK1 with Adi3 was not abolished by either of these Adi3 kinase activity mutants (Fig. 1A).
This was the case whether the proteins were in the bait or prey vectors (Fig. 1A) suggesting that kinase activity is not required for this interaction. The SlSnRK1 and Adi3 interaction was also tested by immunoprecipitation. GST-Adi3 immunoprecipitated with an α -GST antibody was not capable of pulling down MBP, but was capable of pulling down MBP-SlSnRK1 (Fig. 1B, compare lanes 5 and 6).

Adi3 Also Interacts With Two SlSnRK1 β -Subunits.
We also tested if Adi3 could interact with two of the previously identified SlSnRK1 β subunits. First, cDNAs for these two tomato β -subunits, SlGal83 and SlSip1 (Bradford et al., 2003), were cloned. The reported SlGal83 sequence is not a full-length ORF and is missing a portion of the 5' end (Bradford et al., 2003). Thus, we used the tomato EST and genomic databases to identify the remaining 5' end of the SlGal83 sequence and to make sure the  S3B). These results indicated that Adi3 is capable of interacting with several members of the SlSnRK1 complex.
Because Adi3 can interact with SlGal83 and SlSip1 it is possible that Adi3 can phosphorylate these β -subunits. The β -subunits from yeast and mammals are known to be phosphorylated (Mitchelhill et al., 1997;Warden et al., 2001;Mangat et al., 2010), while phosphorylation of the plant β -subunits has not been reported to date. Thus, the ability of Adi3 to phosphorylate the SlGal83 and/or SlSip1 β -subunits was examined. Kinase assays showed that both wild-type Adi3 and constitutively-active Adi3 S539D were able to phosphorylate SlGal83 with Adi3 S539D phosphorylating SlGal83 ~ 6 times more than wild-type ( Fig. 2A, compare lanes 6 and 8).
Interestingly, neither form of Adi3 was capable of phosphorylating SlSip1 ( Fig. 2A, lanes 9-11) even though Adi3 can interact with SlSip1 (Fig. 1B, Fig. S4) and the proteins derived from these ORFs appear to be more related to SlSip1 and the Arabidopsis β -subunit AKINβ2 than to SlGal83 (Fig. 2B). Next, the phosphorylation of Tau1 and Tau2 by Adi3 S539D was tested using in vitro kinase assays, which showed that Adi3 did not phosphorylate Tau1 or Tau2 to a significant level and only phosphorylated SlGal83 (Fig. 2C).
Since Adi3 only phosphorylates SlGal83 and not the other β -subunits we focused on SlGal83, and confirmed that it is a functional SnRK1 β -subunit using yeast complementation that was not done in the initial SlGal83 study (Bradford et al., 2003). In yeast the Snf1 complex functions to allow growth on alternative carbon sources such as sucrose (Carlson et al., 1981;Polge and Thomas, 2007)  confirmation of SlGal83 complementation of sip1Δsip2Δgal83Δ yeast, we tested for restoration of invertase activity, which is regulated by the Snf1 complex under low glucose conditions (Carlson et al., 1984). Our results show that SlGal83-GFP was able to restore basal and low glucose-induced invertase activity to sip1Δsip2Δgal83Δ yeast (Supplemental Fig. S5B). These studies confirm SlGal83 as a true SnRK1 β -subunit and that SlGal83-GFP is functional in vivo.

Identification of Serine 26 as the Adi3 phosphorylation site on SlGal83.
In an effort to identify the SlGal83 residue phosphorylated by Adi3 we carried out a kinase assay screen of several SlGal83 Ser mutants. Within the SlGal83 protein there are 28 Ser amino acids (Supplemental Fig. S2), 17 of which were mutated to Ala and tested for loss of phosphorylation by Adi3 using in vitro kinase assays. Once the assays were completed, the SlGal83 phosphorylation levels were normalized to the SlGal83 and Adi3 protein levels in each assay, and the amount of SlGal83 phosphorylation was expressed as a percentage of wild-type SlGal83 phosphorylation. The results indicate that while many of the mutations slightly increased or decreased the ability of Adi3 to phosphorylate SlGal83, only the Ser26A mutation completely eliminated phosphorylation by Adi3 (Fig. 3A, lane 3). There are 8 Thr residues in SlGal83 (Supplemental Fig. S2). Alanine mutation of one Thr residue did not eliminate Adi3 phosphorylation (data not shown) and the remaining 7 Thr were not tested since Ser26A was a complete knockout of Adi3 phosphorylation of SlGal83 ( phosphorylation and no peptides were found with both Ser26 and Ser30 phosphorylation.

Tomato Gal83 is Phosphorylated in vivo
In order to analyze the in vivo phosphorylation status of SlGal83 we used an alteration to the standard SDS-PAGE by adjusting the ratio of bis-acrylamide to acrylamide. This method has been used to distinguish different phosphorylation states of yeast phosphatidylinositol 4-kinase (Demmel et al., 2008). SlGal83-GFP transgenic Arabidopsis plants were created and the SlGal83-GFP protein analyzed by α -GFP western blot using increasing ratios of bis-acrylamide to acrylamide. The 1:200 bis-acrylamide:acrylamide ratio was capable of separating five different forms of SlGal83-GFP and two of these forms are lost when expressing the SlGal83 S26A -GFP protein (Supplemental Fig. S8). This would suggest that the 1:200 SDS-PAGE/α-GFP western blot can be used to effectively separate and identify different modified forms of SlGal83.
Next, the in vivo phosphorylation status of SlGal83 as expressed in tomato was analyzed.
SlGal83-GFP was expressed in protoplasts, an extract made, the extract treated with λ phosphatase, and SlGal83-GFP analyzed using 1:200 gels/α-GFP western blot. In the presence of λ phosphatase SlGal83-GFP appeared as a single band (

Adi3 Phosphorylates SlGal83 in vivo.
We looked for evidence that SlGal83 Ser26 is phosphorylated in vivo by Adi3 using a coexpression approach. SlGal83-GFP, SlGal83 S26A -GFP, and HA-Adi3 were coexpressed in tomato protoplasts and the banding pattern of phosphorylated SlGal83-GFP analyzed by 1:200 gels/α-GFP western blot. In the absence of HA-Adi3, SlGal83-GFP and SlGal83-GFP S26A appeared as was seen in Fig In order to begin to analyze possible roles for Adi3 phosphorylation of SlGal83 we first utilized the sip1Δsip2Δgal83Δ yeast complementation assay. The ability of the SlGal83 S26A -GFP and SlGal83 S26D -GFP proteins to complement the sip1Δsip2Δgal83Δ cells was tested and the results indicate these proteins complement to an extent similar to that of wild-type SlGal83-GFP (Supplemental Fig. S5A). This suggests Adi3 phosphorylation of SlGal83 may not affect the function, at least in a heterologous system, of controlling growth on alternate carbon sources.
Given the role of Adi3 in suppression of cell death (Devarenne et al., 2006;Ek-Ramos et al., 2010) and that Adi3 can phosphorylate SlGal83, the ability of SlGal83 and its Ser26 phosphorylation mutants to suppress cell death was analyzed in tomato cells. It is known that high levels of NaCl are capable of inducing cell death in plants ( Katsuhara and Kawasaki, 1996;Lin et al., 2006;Tuteja, 2007;Jiang et al., 2008;Affenzeller et al., 2009;Banu et al., 2009;Chen et al., 2009;Wang et al., 2010) and a functional Snf1 complex has been shown to be required for yeast cell survival in the presence of high NaCl (Hong and Carlson, 2007). We expressed SlGal83-GFP, SlGal83 S26A -GFP, SlGal83 S26D -GFP, and GFP-Adi3 in tomato protoplasts, treated them with 200 mM NaCl, and measured cell viability over a 5.5 hr time course. Both Adi3 and SlGal83 were capable of CDS activity and provided increased cell viability in response to NaCl compared to the vector transformed sample (Fig. 5A). The SlGal83 Ser26 phosphorylation mutants did not confer increased or decreased cell viability over wild-type SlGal83 (Fig. 5D).
These results indicated that SlGal83 does have a role in cell death suppression, but phosphorylation of Ser26 may not play a role in controlling SlGal83 CDS activity.
Next, the affect of SlGal83 phosphorylation on SlSnRK1 complex kinase activity was tested.
In order to cary out these assays, an in vitro active SnRK complex must be assembled. Thus, the SlSnRK complex members studied here were analyzed for the formation of an active complex by testing kinase activity against the AMPK/SnRK1 SAMS peptide substrate (HMRSAMSGLHLVKRR; phosphorylation site bold and underlined) (Halford et al., 2003). We also cloned the tomato cDNA for Snf4, which encodes the γ -subunit of the SlSnRK complex (Bradford et al., 2003) for inclusion in the kinase assays. The SAMS phosphorylation (Fig. 5B, column 3). Addition of SlSnf4 marginally, but significantly increased SlSnRK1 T175D SAMS phosphorylation (Fig. 5B, column 4). Inclusion of all complex subunits (SlSnRK1, SlSnf4, SlGal83) imparted a greater increase in SlSnRK1 T175D SAMS phosphorylation (Fig. 5B, column 5). These assays show that the SlSnRK1 subunits comprise a functional complex. To the best of our knowledge, this is the first report of reconstituting an active plant SnRK complex in vitro.
The contribution of SlGal83 Ser26 phosphorylation towards SlSnRK1 kinase activity on the SAMS peptide was analyzed by including the SlGal83 S26D protein in the complex or adding Adi3 to the complex. The results show that SlGal83 S26D conferred a slight yet statistically significant decrease in SlSnRK1 SAMS phosphorylation (Fig. 5B, column 6), while the addition of Adi3 S539D to the assay drastically lowered the phosphorylation of SAMS to a level close to that of SlSnRK1 alone (Fig. 5B, column 7). This drop in SAMS phosphorylation appears to partially depend on Adi3 kinase activity as inclusion of the kinase-inactive Adi3 K337Q restored activity of the complex similar to SlSnRK1 T175D alone, but not to the level of the full complex (Fig. 5B, column 8). This would suggest that even though Adi3 does not phosphorylate SlSnRK1 ( Fig.   2A, lane 14), it may inhibit SnKR1 kinase activity through their interaction. This appears to be the case since kinase-active Adi3 S539D or kinase-inactive Adi3 K337Q reduced SAMS phosphorylation by SlSnRK T175D and SlSnRK T175D + Snf4 close to the level of SlSnRK1 alone (Fig. 5B,columns 9,10,11,12). In order to analyze if the drop in complex kinase activity in the presence of Adi3 is due to an additional protein in the assay, the analysis was repeated with the addition of GST protein. This assay had strong kinase activity, but not to the level of the full complex (Fig. 5B, column 13). This would suggest that some loss of kinase activity in the presence of Adi3 could be due to the addition of an additional protein. To take this into account, the values in Fig. 5B were normalized to that of the assay in the presence of GST; i.e the GST sample was set as 100% and the other samples were expressed as a percentage of this value. Fig.   5C shows that when expressed in this manner, the trends do not change.
We extended the SnRK1 SAMS phosphorylation assays to a more in vivo approach by expressing SlGal83-GFP or SlGal83 S26D -GFP in tomato protoplasts, making extracts of these cells, and using the extract to phosphorylate the SAMS peptide. We found that the extract from SlGal83 S26D -GFP expressing cells had greatly reduced SAMS phosphorylation compared to expression of SlGal83-GFP (Fig. 5D). This reduction in SAMS phosphorylation is much lower than what was seen for the in vitro assay (Fig. 5B, C) suggesting that a more in vivo context is needed to better realize the effects of Ser26 phosphorylation. Taken together, these kinase assay data suggest that the Adi3 interaction with the SlSnRK complex has the ability to inhibit the kinase activity of the complex. This may be mediated through two mechanisms, phosphorylation of SlGal83 and interaction with SlSnRK1.

DISCUSSION
In the present study we present evidence for the interaction of Adi3 with the SnRK complex in tomato. Our finding that Adi3 can only phosphorylate the Gal83 SnRK β -subunit out of the four β -subunits identified in tomato has far-reaching implications since Snf1/AMPK/SnRK1 β subunits control cellular localization and substrate specificity of the complex (Mitchelhill et al., 1997;Vincent and Carlson, 1999;Vincent et al., 2001Vincent et al., , 2001Warden et al., 2001). Additionally, β -subunit phosphorylation has been associated with regulation of some of these β -subunit functions (Mitchelhill et al., 1997;Warden et al., 2001;Hedbacker et al., 2004;Mangat et al., 2010), and the SnRK complex appears to link signaling connected with metabolism and stresses (Halford and Hey, 2009;Hey et al., 2010). Given the role of Adi3 in cell death control our studies add additional evidence for the connection of SnRK with stress signaling. Alternatively, Adi3 may also be involved in the direct regulation of metabolism through its interactions with the SlSnRK1 complex.

A Role for Adi3 Phosphorylation in Regulating SnRK Complex Kinase Activity?
We have shown that Adi3 phosphorylates SlGal83 at Ser26 (Fig. 2)  of SlGal83 S26D was seen in vivo (Fig. 5D), but was much less drastic in vitro (Fig. 5C) suggesting there is an in vivo role for Ser26 phosphorylation in controlling SnRK1 kinase activity.
Interestingly, the restoration of SAMS phosphorylation when including kinase-inactive Adi3 K337Q would suggest that Adi3 kinase activity is at least partially required for this large inhibition of SnRK1 activity in vitro and may suggest additional Adi3 phosphorylation sites on SlGal83 for controlling activity. It is possible that Ser30 is one of these sites since we identified Our results also suggest that the interaction of Adi3 with SlSnRK1 is capable of suppressing SlSnRK1 kinase activity (Fig. 5B). In the absence of SlGal83 the kinase-active or -inactive forms of Adi3 are capable of suppressing SlSnRK1 kinase activity (Fig. 5B). This apparently contradicts the finding that the kinase-inactive Adi3 can restore activity of the complex in the presence of SlGal83. However, these results may indicate that the Adi3/SlGal83 interaction affects the ability of Adi3 to fully interact with and inhibit SlSnRK1. Eliminating SlGal83 from the assay would then allow for full interaction between Adi3 and SlSnRK1 and stronger activity inhibition. The interaction of Adi3 with SlSnRK1 may be inhibiting the ability of SlSnRK1 to bind the SAMS substrate or even ATP. These data also help to explain the detection of SlSnRK1 as an Adi3 Y2H interactor even though Adi3 does not phosphorylate SlSnRK1 as well as shed light on the biological significance for this interaction. Given the role of Adi3 in the host response to Pst and the function of SnRK1 in stress signaling, Adi3 may be directing reallocation of cellular energy reserves by modulating SlSnRK1 kinase activity during the resistance response of tomato to Pst. Studies using Nicotiana attuneata show that photosynthate is reallocated to the roots in response to herbivore attack through the down-regulation of SnRK

Multiple Roles for β -Subunit Phosphorylation.
Snf1/AMPK/SnRK1 β -subunits appear to be phosphorylated on several amino acids and our studies also support phosphorylation at several residues on SlGal83. Expression of SlGal83-GFP in plant cells showed the existence of multiple phosphorylated protein bands based on our λ phosphatase treatments, one of which contains Ser26 phosphorylation ( Fig. 4A et al., 2004). However, mutation of 4 potential PKA phosphorylation sites did not affect ScSip1 cellular localization (Hedbacker et al., 2004).
Taken together it appears that the role of Snf1/AMPK/SnRK1 β -subunit phosphorylation is not fully understood and will be an important area of research for the future. From our studies the full role of SlGal83 Ser26 phosphorylation by Adi3 is not clear. It appears to have only a minor role in controlling SlSnRK1 complex kinase activity. So, additional functions attributable to this phosphorylation event will be important to identify in the future. Given the role of β subunits in controlling Snf1/AMPK/SnRK1 complex localization and phosphorylation playing a role in this function, it will be important to examine the contribution of phosphorylation by Adi3 in controlling SlGal83 cellular localization. Consequently, the full extent of the SlGal83 Ser26 phosphorylation event by Adi3 remains to be determined.

Is There a Link Between Cell Death Control and Metabolism?
An important aspect of PCD is the reallocation of cellular resources such as proteins and sugars. This is particularly true of the cell death that occurs during leaf senescence (van Doorn andWoltering, 2004, 2008;Guiboileau et al., 2010). In fact, the reuse of cellular materials was suggested as early as 1891 from the examination of cell death associated with xylem development (Lange, 1891). Thus, it may not be surprising that a gene controlling PCD would also be able to regulate how cells utilize and/or mobilize energy sources. This appears to be the case for mammalian PKB. While it is well known that PKB suppresses cell death by phosphorylating and inactivating proapoptotic proteins or activating antiapoptotic proteins (Luo et al., 2003;Carnero, 2010), PKB also functions in the regulation of metabolism through the control of glycolytic enzymes and glucose uptake (Plas and Thompson, 2002;Carnero, 2010 AMPK activity. While PKB and AMPK do not directly interact with each other, there is substantial crosstalk between the pathways. For example, activation of PKB has been shown to down regulate AMPK activity and thus a decrease in AMP/ATP cellular ratios (Kovacic et al., 2003;Hahn-Windgassen et al., 2005). Conversely, activation of AMPK has been shown to inactivate PKB-regulated glycolysis (Grabacka and Reiss, 2008). Combining our current and previous Adi3 studies raises the possibility that Adi3 functions similarly to PKB in cell death and metabolism control. Further studies on the role of Adi3 association with the SlSnRK1 complex, especially phosphorylation of Gal83, will be required to fully understand if there is a connection between Adi3-mediated cell death control and SlSnRK1 metabolism control. SnRK1, Gal83, Sip1, Tau1, Tau2, and Snf4.

Cloning of Tomato
All primers and restriction sites used in this study for ORF amplification, cloning, and mutagenesis are listed in Supplemental Table S1 and the primers used to amplify all genes were designed using sequence data obtained from the Sol Genomics Network (SGN) databases (http://solgenomics.net/). The ORFs for SlSnRK1, SlSip1, SlSnf4, SlTau1, and SlTau2 were obtained by RT-PCR using cDNA generated with Superscript III (Invitrogen) from tomato total RNA isolated from 4-week-old leaves. Primers used to amplify SlSnRK1 (accession #AF143743) were based on the unigene SGN-U564382. The cDNA for SlSip1 (accession #AF322108) in unigene SGN-U575258 and reported in Bradford et al. (2003) appeared to lack a portion of the 5' end of the cDNA when compared to homologous β -subunits from yeast and The SlSnf4 ORF (accession #AF419320) was amplified by PCR using the published sequence (Bradford et al., 2003). Mutagenesis of SlSnRK1 and SlGal83 was performed using Pfu Turbo Polymerase (Stratagene) and the primer pairs listed in Supplemental Table S1. Cloning of Adi3 and its kinase activity mutants were described previously (Devarenne et al., 2006).

Recombinant Protein Expression and Purification.
The ORFs for SlSnRK1, SlGal83, and SlSip1 were cloned as N-terminal MBP fusions into pMAL-c2 vector (New England Biolabs). Recombinant proteins were expressed in E. coli BL21 Star (DE3) as described previously (Devarenne et al., 2006) and purified using maltose binding resin (New England Biolabs) manufacturer protocols. For GST-Adi3, the Adi3 ORF was cloned into the pGEX-4T N-terminal GST fusion vector (GE-Healthcare) and protein was expressed and purified as recommended by the manufacturer. After elution, all fusion proteins were concentrated using Amicon Ultra centrifugal filters (Millipore) and added to buffer for final concentrations of 50% glycerol, 50 mM Tris-HCl pH7.5, 0.5 mM EDTA, 100 mM NaCl.
Protein concentrations were quantified using Bio-Rad Protein Assay Kit before storage at -20C.

Yeast Two-Hybrid Assay
Y2H assays where conducted using pEG202 for the bait vector and pJG4-5 for the prey vector as described previously (Devarenne et al, 2006). Constructs were transformed into yeast strain EGY48 containing the pSH18-34 reporter vector and analyzed for LacZ gene expression on X-Gal containing plates. Protein expression was confirmed by western blot. All other procedures for the Y2H assays and Y2H library screen for identifying Adi3 interactors followed standard procedures as previously described (Golemis et al., 2008).

Yeast Complementation and Invertase Assays
The ORF for SlGal83 and its Ser26 mutants were fused to a C-terminal eGFP tag under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter in the modified vector MBB263. The yeast β -subunit knockout strain MCY4040 (MATα sip1Δ::KanMX6 sip2Δ3::LEU2 gal83::TRP1 his3-Δ200 leu2-3,112 trp1Δ1 ura3-52 lys2-801) (Vincent et al., 2001) was transformed with the SlGal83 constructs using the standard lithium acetate/PEG method. Transformants were screened on plates of complete minimal (CM) media with 2% glucose and lacking leucine, tryptophan, and uracil. Recovered colonies were grown in liquid CM 2% glucose medium for 48 hrs and 5-fold serial dilutions were spotted on selective media supplemented with either 2% glucose or 2% sucrose and incubated at 30˚C for 2 days (2% glucose) or 6-7 days (2% sucrose). Invertase assays were performed as previously reported (Celenza and Carlson, 1989;Bradford et al., 2003). Invertase activity of derepressed (0.05% glucose) and glucose-repressed (2% glucose) cells was estimated as a measure of the amount of sucrose metabolized into glucose using the Glucose (GO) Assay kit (Sigma) as described by the manufacturer.

Pull Down Assays
Immobilized glutathione beads (Thermo Scientific) were equilibrated by washing three times with 200 μ l of binding buffer (50 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 5 mM EDTA). For each pull down 1 μ g of either GST or GST-Adi3 and equivalent protein amounts of MBP, MBP-Gal83, MBP-Sip1, and MBP-SnRK1 were mixed in a final volume of 30 μ l.
Samples were incubated for 15 min at room temperature followed by addition of buffer preequilibrated glutathione resin to each sample and incubation for 1 hr at 4˚C on an orbital shaker.
The resin with bound proteins was pelleted by centrifugation at 100 x g and washed 5 times with 200 μ l of wash buffer (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 5 mM EDTA). Bound proteins were eluted using 1x SDS-PAGE sample buffer, resolved by 12% SDS-PAGE, and analyzed by western blotting using α -GST (Santa Cruz Biotechnology) at 1:15,000 and α -MBP (New England BioLabs) at 1:5,000 for pull downs or 1:10,000 for loading controls. to be slightly stronger using MnCl 2 and therefore, was used for all SlSNRK1 autophosphorylation assays. However, SlSnRK1 substrate phosphorylation was comparable using MnCl 2 or MgCl 2 as a cofactor. Therefore, MgCl 2 was used for all SlSnRK1 substrate phosphorylation experiments. Adi3 substrate phosphorylation assays contained 5 μ g of purified MBP-Adi3 or MBP-Adi3 S539D and 2 μ g of MBP-Gal83, MBP-Gal83 mutants, or MBP-Sip1. For SAMS peptide (HMRSAMSGLHLVKRR) phosphorylation assays were performed as described previously (Davies et al., 1989). Assay conditions for SlSnRK1 phosphorylation of the SAMS peptide were as for the SlSnRK1 substrate phosphorylation assays above plus 100 μ M SAMS peptide (AnaSpec). Reactions were spotted on phosphocellulose p81 paper (Whatman), washed three times in 1% H 3 PO 4 , once in acetone, the paper dried, and the incorporated radioactivity counted using a Beckman LS5000TA scintillation counter. For SAMS phosphorylation with protoplast lysates, 4 x 10 5 tomato protoplasts expressing empty pTEX vector, SlGAL83-GFP, and SlGAL83 S26D -GFP were lysed by vortexing in a buffer containing 50mM Tris pH8.0, 1mM EDTA, 50mM NaCl, 8% Glycerol, 5mM DTT, 2% plant protease inhibitor cocktail (Sigma) and 2% plant phosphatase inhibitor cocktail (Sigma). Extracts were cleared by centrifugation at 4°C, 13,000 g for 10 minutes. Protein concentration was estimated as described above and lysates were adjusted to equal protein concentrations with lysis buffer.
Reactions were done as described above, but using a buffer containing 40 mM HEPES-KOH pH  phosphorylated peptides were manually inspected to ensure confidence in phosphorylation site assignment.

Phosphatase Treatment
Gal83-GFP proteins were exercised in tomato protoplasts from pTEX for 22 hrs and were lysed in ice-cold extraction buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 2 mM DTT, 2.5% plant protease inhibitor cocktail (Sigma), and 6 μ M epoxymicin (Enzo Life Sciences). Lysates were split into two fractions; one for phosphatase treatment and one for a no treatment control. Both fractions were adjusted to 3 mM MnCl 2 in λ phosphatase buffer (50mM HEPES pH7.5, 100mM NaCl, 2mM DTT, 0.01% Brij-35) in a final volume of 100 μ l. The no treatment fraction was additionally adjusted to 2% phosphatase inhibitors (Sigma, phosphatase inhibitor cocktail 1). Reactions were started with the addition of 800 units of λ phosphatase (New England BioLabs), incubated at 30˚C for 30 min, and reactions terminated by addition of 1x SDS-PAGE sample buffer. Samples were then resolved by 7.5% SDS-PAGE with a 1:200 bis-acrylamide :acrylamide ratio and analyzed by α -GFP western blotting.

Protoplast Protein Expression and Cell Death Assays
The Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers JF895513 (SlGal83), JF8955212 (SlSip1), JQ846034 (SlTau1), JQ846035 ) SlTau2)

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Alignment of SnRK proteins from tomato and Arabidopsis.