SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew

Abstract

Plants recognize pathogens and activate immune responses, which usually involve massive transcriptional reprogramming. The evolutionarily conserved kinase, Sucrose non-fermenting-related kinase 1 (SnRK1), functions as a metabolic regulator that is essential for plant growth and stress responses. Here, we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library. SnRK1 interacts with WRKY3 in yeast, as confirmed by pull-down and luciferase complementation assays. Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus. Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus. Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation. Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor, and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein. SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh. Transient expression of SnRK1 reduces fungal haustorium formation in barley cells, which probably requires SnRK1 nuclear localization and kinase activity. Together, these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor, revealing a new regulatory mechanism of immune derepression in plants.

Keywords: SnRK1; WRKY transcription factor; immunity; phosphorylation; powdery mildew fungus.

Figure 1

WRKY3 Is a Suppressor of Barley Disease Resistance to B. graminis Fungus. (A) Transient overexpression of WRKY3 in barley epidermal cells represses basal and MLA1-mediated isolate-specific disease resistance against powdery mildew fungus. Haustorium index was scored at 48 h after spore inoculation in leaf epidermal cells expressing the empty vector (EV) or the indicated plasmids in the MLA1-containing near-isogenic line P01. Bgh isolates A6 and K1 were used for inoculation. At least 50 cells were analyzed in each experiment, and the data show average values calculated from three independent experiments. Data represent the means ± SD from three independent replicates. ∗∗P < 0.01, significant difference between the EV control and WRKY3 using Student’s t-test. (B) Silencing of WRKY3 using BSMV-VIGS enhances basal disease resistance against powdery mildew fungus in barley. Bgh microcolony index was scored at 48 h after spore inoculation with isolate A6. The ±SD analyses and error bars reflect the means of three independent experiments. At least 2000 interaction sites were analyzed under the microscope in one experiment. Data represent the means ± SD from three independent replicates. ∗P < 0.05, significant difference using Student’s t-test. (C) Stable overexpression of WRKY3 in transgenic barley compromises disease resistance to powdery mildew fungus. Representative images show the effects of WRKY3 overexpression in barley against powdery mildew infection at the seedling stage. Western blotting confirms the presence of WRKY3-HA accumulation in transgenic GP plants. Scale bar, 5 mm. (D) Statistical analyses of microcolony formation rate in transgenic GP lines overexpressing WRKY3. Bgh microcolony index was scored at 48 h after inoculation with isolate K1. The ±SD analyses and error bars reflect the means of three independent experiments. At least 2000 interaction sites were analyzed under the microscope in one experiment. Data represent the means ± SD from three independent replicates. ∗P < 0.05, significant difference using Student’s t-test. (E) Representative images showing microcolony formation on the leaf surface of transgenic plants and recipient GP plants. Scale bar, 200 μm.

Figure 2

SnRK1 Interacts with WRKY3. (A) SnRK1 interacts with WRKY3 in yeast. Full-length and truncated versions of WRKY3 were individually fused to the DNA-binding domain (BD) in pLexA. SnRK1 was fused to the activation domain (AD) in pB42AD. (B) SnRK1 interacts with WRKY3 in MBP pull-down assays. The recombinant MBP-WRKY3 and SnRK1-3xHA expressed in N. benthamiana were subjected to MBP pull-down analysis. MBP alone served as a negative control. The interacting protein was revealed by anti-HA immunoblotting. The experiment was repeated three times with similar results. (C) SnRK1 interacts with WRKY3, demonstrated by split-luciferase assays. The N- or C-terminal fragment of LUC (nLUC or cLUC) was fused with the indicated proteins. The indicated constructs were coexpressed in N. benthamiana by agroinfiltration. Images of chemiluminescence were recorded by applying 0.5 mM luciferin 36 h after infiltration. Similar results were obtained in three biological replicates. (D) SnRK1 interacts with WRKY3, indicated by FLIM–FRET analysis. A CFP fluorescence lifetime image of the nucleus of a representative cell expressing the indicated proteins is shown. The average fluorescence lifetime obtained for each pixel is encoded by color as indicated by the scale. Scale bar, 10 μm. (E) SnRK1 interacts with WRKY3, indicated by FLIM–FRET analysis. A CFP fluorescence decay frequency curve was measured for nuclei expressing the indicated proteins.

Figure 3

SnRK1 Phosphorylates WRKY3 Mainly at S83 and S112. (A) Analysis of phosphorylation sites of WRKY3 by activated SnRK1 in vitro. The analysis was performed by LC–tandem mass spectrometry (MS/MS) (see Methods). Black bars represent the sequence coverage enriched with each proteolytic enzyme. Asterisks mark each identified phosphorylation site. Residue S112 (red) of WRKY3 resides in an SnRK1 phosphorylation consensus sequence, as shown below. The conserved domains of WRKY3 were labeled as follows: LxLxLx (red box), putative repression motif; LZ (yellow box), leucine zipper; NLS (blue box), nuclear localization signal; WRKY (green box), WRKY DNA-binding motif. (B) SnRK1 phosphorylates WRKY3 mainly at S83 and S112. His-tagged WRKY3 with or without mutations at S83 or S112 was incubated with SnRK1 kinase (SnRK1-KD) and GRIK1 and analyzed by Phos-tag mobility shift assays. A red asterisk marks each shifted band. Black arrows indicate single and double phosphorylated WRKY3 derivatives. Red arrows indicate non-phosphorylated WRKY3 proteins.

Figure 4

Phosphorylation at S83 and S112 Destabilizes WRKY3. (A) 3xHA-tagged WRKY3 with or without S/A substitution at S83 or S112 was transiently coexpressed with YFP-tagged SnRK1 and Myc-tagged GRIK1 in N. benthamiana leaves by agroinfiltration. Protein accumulation was examined by western blotting at 3 days after infiltration. Numbers below the blots indicate the relative protein levels calculated with ImageJ software. MG132 was applied 12–14 h before sample harvesting. DMSO was used as a mock control, and actin served as a loading control. (B) Accumulation of WRKY3-3xHA was detected in transgenic barley lines in the absence or presence of Bgh infection. The addition of MG132 inhibited the degradation of WRKY3 in vitro.

Figure 5

WRKY3^S83A and WRKY3^S112A Mutants Retain Suppressor Functions. (A) WRKY3 with an S-to-A substitution at S83 or S112 retains transcriptional repressor activity. A LUC reporter containing the 5×GAL4 UAS (upstream activating sequence), effectors fused with the GAL4 DNA-binding domain (DBD), and an internal control were cotransfected into Arabidopsis protoplasts at a ratio of 6:6:2. DBD alone and DBD fused with the strong activator VP16 were used as negative and positive controls, respectively. (B) WRKY3 with an S-to-A substitution at S83 or S112 retains its defense repressor activity against Bgh in barley. WRKY3 or its phosphorylation-null mutant plasmids were coexpressed with the GUS reporter gene in barley leaf epidermal cells of the MLA1-containing near-isogenic line P01 by particle bombardment delivery. The virulent Bgh isolate A6 containing virMLA1 was used for inoculation. The haustorium index was scored at 48 h after spore inoculation. At least 50 cells were analyzed in one experiment, and the data shown are calculated from three independent experiments. Data represent the means ± SD from three independent replicates. ∗∗P < 0.01, significant difference using Student’s t-test.

Figure 6

SnRK1 Positively Regulates Barley Disease Resistance to the B. graminis Fungus. (A) Overexpression of SnRK1 or SnRK1^K139R enhances or compromises basal disease resistance against powdery mildew fungus in barley, respectively. Representative images of seedling leaves from GP and transgenic plants were taken at 7 days after inoculation with Bgh K1. Protein accumulation of SnRK1 and SnRK1K139R was confirmed by western blotting. Scale bar, 5 mm. (B) Overexpression of SnRK1 or SnRK1^K139R decreases or increases the microcolony formation rate on barley leaf surfaces, respectively. The Bgh microcolony index was scored at 2 days after inoculation with Bgh K1. Three independent experiments were performed, and similar results were obtained. At least 2000 interaction sites were analyzed under the microscope in each experiment. Data represent the means ± SD from three independent replicates. ∗P < 0.05 and ∗∗P < 0.01, significant difference using Student’s t-test. (C) Representative images of microcolonies on the leaf surface of transgenic barley overexpressing SnRK1 or SnRK1^K139R. Scale bar, 200 μm.

Figure 7

SnRK1 Nuclear Localization Is Essential for Disease Resistance. (A) Subcellular localization analysis of SnRK1 fusion proteins. SnRK1-YFP-NLS, SnRK1-YFP-nls, SnRK1-YFP-NES, and SnRK1-YFP-nes were expressed in N. benthamiana leaves by agroinfiltration. The confocal images were taken at 48 h after infiltration. Scale bars, 50 μm. (B) Analysis of disease resistance activity of indicated SnRK1 fusion proteins against Bgh infection. The haustorium index was scored at 48 h after spore inoculation in the leaf epidermal cells of barley cultivar P01 expressing the indicated plasmids. The experiment was repeated independently three times, and similar results were obtained.