The Sucrose non-fermenting related kinase 1 (SnRK1) complex is a hetero trimer found in plants consisting of a catalytic a-subunit and regulatory b and g subunits. The SnRK1 complex can regulate a variety of metabolic pathways in response to changes of energy status in the cell. Furthermore, the SnRK1 complex was proposed to play a role in bacterial resistance due to its interaction with the programmed cell death (PCD) inhibitor Adi3 (AvrPto-dependent Ptointeracting protein 3). In the presence of bacteria expressing the effector protein AvrPto, Adi3 will not be phosphorylated, thus releasing its cell death suppression activity and PCD occurs. PCD signaling components may regulate SnRK1 complex activity, the SnRK1 complex in turn can regulate downstream metabolic pathways through phosphorylation of key enzymes in the metabolic pathways or transcription reprogramming. In order to better understand the function and regulation of the tomato SnRK1 complex, the activity and regulation of the a-subunit was studied. A previous study identified the SnRK1.1 asubunit. A search of the tomato genome database resulted in the identification a second SnRK1 a-subunit, SnRK1.2, and the upstream activation kinase SnAK. In the phylogenetic analysis of SnRK1 sequences from monocots and dicots SlSnRK1.2 clusters only with other Solanaceae SnRK1.2 sequences, suggesting possible functional divergence of these kinases from other SnRK1 kinases. Tomato SnRK1.2 exhibits lower kinase activity compared to SnRK1.1 even after SnAK activation. Moreover, in vitro reconstitution of the SnRK1 complex revealed that SnRK1.2 complexes could have higher activity if Sip1 or Tau2 was used as the b-subunit. On the other hand, SnRK1.1 have higher activity when Gal83 or Tau2 was used as the b-subunit. These studies suggest the SnRK1.2 phylogenetic divergence and lower SnRK1.2 kinase activity compared to SnRK1.1 may be indicative of different in vivo roles for each kinase. One substrate of SnRK1, nitrate reductase (NR), was used to study the effect of Adi3 phosphorylation status on NR activity through the regulation of SnRK1 complex. Contrary to previous observations that Adi3 inhibits SnRK1 activity, our result shows that a phosphomimetic version of Adi3 caused an increase in NR phosphorylation by SnRK1.1. While there is previous contradicting evidence for both promotional and inhibitory effects of the ?-subunit on NR, the results here show that addition of the Gal83 ?-subunit had no effect on NR activity. Finally, we studied the effect of effector proteins, AvrPto and AvrPtoB, on SnRK1 complex activities. Our results show that AvrPto inhibits SnRK1 activity in resistant plants, while it had no effect on susceptible plants.
