Mechanism Of The Interaction Between TYLCCNB βC1 And Tomato Host Factor SlSnRK1

Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite geminivirus, associated with a betasatellite molecule (TYLCCNB), which is essential for the induction of typical disease symptoms in natural hosts. TYLCCNB encode a protein, known asβC1, in the complementary-sense orientation. TYLCCNBβC1 is a pathogenicity determinant and a suppressor of RNA silencing. To better understand the molecular basis ofβC1 in the pathogenicity, the interaction between TYLCCNBβC1 and tomato host factors was investigated in this study.A yeast two-hybrid screen was performed to identify host proteins from a tomato cDNA library that interact with TYLCCNBβC1, using the TYLCCNBβC1 as bait. The plasmid pGBKT7-βC1, tomato cDNA and the linear vector pGADT7-Rec were co-transformed into Saccharomyces cerevisiae strain AH109, and the double transformants were assayed for histidine prototrophy and a-galactosidase activity. One cDNA clone designated as SlSnRKl was selected, and its interaction withβC1 was further confirmed using yeast two-hybrid system. Bimolecular fluorescence complementation (BiFC) vectors pβC1-YFPN and pSlSnRK1-YFPc were constructed, and BiFC assay showedβC1 interacted with SlSnRKl in living Nicotiana benthamiana cells.The full-length cDNA of the SlSnRK1 has 1545 nucleotides encoding a protein of 514 amino acid (58,824 kD). SlSnRK1 is closely related to SnRK1 in N. benthamiana, NPK5 in N. tabacum, and AKIN11 in Arabidopsis thaliana (95%,87% and 78%, respectively). Putative functional domains in SlSnRKl were deduced from InterProScan online software (http://www.ebi.ac.uk/Tools/InterProScan/). SlSnRKl contains a conserved kinase domain (KD), a internal ubiquitin associated domain (UBA) and an auto-inhibitory sequence (AIS) domain, as well as a C-terminal domain that responsible forβsubunit binding and formation of SnRK1 complex (CTD). To determine the subcellular localization of SlSnRKl protein, SlSnRKl was tagged at its N terminus with GFP by inserting PCR-amplified SlSnRKl cDNAs into the plasmid pCHF3-GFP to produce the expression vector pCHF3-GFP-SlSnRKl. Distribution of GFP-tagged SlSnRKl within cytoplasm and nucleus was observed by confocal microscopy in N. benthamiana leaves infiltrated with Agrobacterium tumefaciens containing the fusion protein of SlSnRKl with GFP.Semi-quantitative RT-PCR and real-time quantitative reverse transcription (RT)-PCR were performed with SlSnRKl specific primers, using total RNA from various tomato tissues as template. Highest accumulation level of SlSnRKl mRNA was found in the flower with intermediate in leaf, and low in root and stem. Real-time quantitative RT-PCR was also performed to compare SlSnRKl transcript accumulation level in infected and mock-inoculated tomato leaves. The results suggest that TYLCCNV/TYLCCNB infection stimulates the accumulation of the SlSnRKl mRNA at early stage of infection, and has no obvious effect in period of the typical symptom appearance, but down-regulate SlSnRKl gene expression at late stage of infection.TYLCCNV/TYLCCNB was inoculated onto wild-type N. benthamiana plants or transgenic N. benthamiana plants expressing Arabidopsis sense SnRKl (S-5). As judged by symptom appearance, transgenic lines S-5, which over-expresses SnRKl, reduced the efficiency of virus infection in comparison with wild-type N. benthamiana. Symptom appearance in transgenic lines S-5 was delayed in comparison with wild-type N. benthamiana, indicating that SlSnRKl is involved in a component of plant innate defense.Yeast plasmids expressing PCI, SlSnRKl and SlSnRKl kinase-dead mutant (SlSnRKlK48R) were constructed. The yeast snfl deletion strain△snfl BY4741 was used to perform the yeast complementation assay and investigate whetherβC1 can impact SlSnRKl kinase activity in yeast cells. In initial experiments, SlSnRKl or SlSnRKlK48R was separately expressed in△snfl BY4741. The results show that SlSnRKl can functionally complement SNF1 in yeast, but kinase-dead mutant (SlSnRKlK48R) losts this function, suggesting SlSnRKl kinase activity is essential for complementation. In subsequent experiments, SlSnRKl andβC1 were co-expressed in△snfl BY4741, the result indicated thatβC1 expression can not inhibit SlSnRK1 activity.To inverstigate the mechanisms of interaction between TYLCCNBβC1 and SlSnRK1, the full-lengthβC1 as well as a truncated SlSnRK1 (named SlSnRKl-KD, which containing the kinase and UBA domain) were separately fused to GST, expressed and purified from Escherichia coli cells, then used in in vitro kinase assays. The autoradiography result indicated thatβC1 can be specifically phosphorylated by SlSnRK1-KD, suggesting the TYLCCNBβC1 protein is a substrate for SlSnRK1 kinase. To find which residues inβC1 serve as the potential target sites for SlSnRKl phosphorylation, the full-length sequence ofβC1 protein was submitted to NetPhos online server for potential phosphorylation sites prediction. Analysis results indicate that two residues ofβC1, the threonine residue at position 78 (T-78) and serine residue at position 33 (S-33), are possible phosphorylation sites for serine/threonine kinases. Therefore sixβC1 point mutants, in which S-33 or T-78 were individually or simultaneously substituted by alanine residues or by aspartate residues, were generated and expressed as GST fusion proteins, and used in in vitro kinase assays. Autoradiography results indicated that mutations on either S-33 or T-78 all significantly impaired the phosphorylation level ofβC1 when compared with wild-typeβC1, suggesting that both S-33 and T-78 ofβC1 are phosphorylation sites for SlSnRK1. Furthermore, the reduction of radioactive intensity caused by mutations on T-78 were less than that observed for S-33, indicating that the S-33 is a preference site for recognition and phosphorylation by SlSnRK1.Alanine and aspartate substitution mutations were introduced into theβC1 gene at the S-33 and/or T-78, and sixβC1 mutant constructs were generated. Infectious clones of mutants and wild type TYLCCNB were used to inoculate N. benthamiana plants together with TYLCCNV. Reduction in the efficiency of virus infection associated with mild symptoms was observed in plants inoculated with phosphorylation-mimic mutants, suggesting that phosphorylation ofβC1 resulted in attenuation of symptoms and reduced the efficiency of viral infection.The above results obtained in this study uncovered a novel role for SlSnRK1 in plant defense response by phosphorylating theβC1 protein.