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

Abstract

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.

Figure 1.

A, Interaction between SlSnRK1 and TYLCCNB-βC1 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)/−His/−Leu/−Trp 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 β-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.]

Figure 2.

BiFC visualization of interaction between TYLCCNB-βC1 and SlSnRK1 in N. benthamiana leaves. A, YFP fluorescence. B, Bright field. C, YFP/bright field overlay. Bars = 50 μm.

Figure 3.

Subcellular localization of SlSnRK1 in N. benthamiana epidermal cells. Micrographs showing cells expressing GFP (top row) or GFP:SlSnRK1 (bottom row) were examined under fluorescence (left), bright field (middle), or an overlay of bright and fluorescence illumination (right) by confocal microscopy. Arrows indicate nuclei. Bars = 50 μm.

Figure 4.

SlSnRK1 mRNA levels in various tomato tissues (A) or TYLCCNV/TYLCCNB- and TYLCCNV-infected plants at 3 DPI (B). Relative mRNA levels in tomato tissues were normalized using EF-1α 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).

Figure 5.

Identification of the binding domains responsible for the SlSnRK1-TYLCCNB-βC1 interaction. A, Schematic representation of the truncated mutants of SlSnRK1 and yeast two-hybrid analysis of their interactions with βC1. The yellow box represents the KD, the gray box represents the UBA domain, the light blue box represents the AIS, and the red box represents the CTD. B, Diagram of deletion mutants of βC1 used to determine the binding requirements for SlSnRK1. The Ser residue at position 33 is indicated as a dark blue box and the Thr residue at position 78 is labeled as a green box. [See online article for color version of this figure.]

Figure 6.

TYLCCNV/TYLCCNB infectivity efficiency and viral DNA accumulation levels in wild-type (WT) and transgenic plants expressing antisense SnRK1 (AS-12) or sense SnRK1 (S-5). A, Course of infection in AS-12, S-5, and wild-type lines. Values represent percentages of systemically infected plants at different DPI. B, DPI 50% in infected AS-12, S-5, and wide-type plants. Different lowercase letters above the bars denote significant differences (Fisher’s lsd method; P < 0.05). All data represent means ± sd of triplicate experiments. In each experiment, 12 plants of each line were inoculated with the A. tumefaciens strain EHA105 culture containing TYLCCNV and TYLCCNB. C, TYLCCNV and TYLCCNB DNA levels in wild-type and transgenic plants expressing antisense SnRK1 or sense SnRK1 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. [See online article for color version of this figure.]

Figure 7.

SlSnRK1 functionally complements SNF1 in yeast (A), and TYLCCNB-βC1 does not inhibit SlSnRK1 activity in yeast (B). Cells of Δsnf1 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). βC1 was expressed from a high-copy plasmid (SCL-βC1), and SlSnRK1^K48R was expressed from a low-copy plasmid (CTU-SlSnRK1^K48R). Δsnf1 BY4741 transformed with the empty vector pESC-Ura or cotransformed with pESC-Ura and SCL-βC1 was used as a negative control. Δsnf1 BY4741 cotransformed with SCU-SlSnRK1 and pESC-Leu served as a positive control. YNB, Yeast nitrogen base. [See online article for color version of this figure.]

Figure 8.

In vitro phosphorylation of βC1. A, SlSnRK1 can specifically phosphorylate βC1. Coomassie blue-stained SDS-PAGE gels (12%; top panel) and the corresponding autoradiograph images (bottom panel) are shown. Due to the similar molecular masses, GST-SAMS (approximately 27 kD) comigrates with GST (approximately 26 kD) during electrophoresis. B, SlSnRK1 phosphorylates βC1 primarily at Ser-33 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 ± se of three replicate experiments. Different lowercase letters above the bars denote significant differences (Fisher’s lsd method; P < 0.05).

Figure 9.

Effects of βC1 phosphorylation site mutations on virus infection and relative viral DNA accumulation levels in agroinoculated N. benthamiana plants. A, Symptoms of plants agroinoculated with TYCCNV and TYCCNB or its mutants (TYLCCNB-S33A, TYLCCNB-S33D, TYLCCNB-T78A, TYLCCNB-T78D, TYLCCNB-S33A/T78A, and TYLCCNB-S33D/T78D) at 17 DPI. CK− 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 ± 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 ± 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.