OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus

Abstract

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Keywords: AvrPib; Importin α; Magnaporthe oryzae; Nuclear localization signal; Oryza sativa; WRKY.

Fig. 1

Interaction of OsIMα1a and OsIMα1b with OsWRKY76. a Schematic diagrams of OsWRKY76.1 (W76.1), OsIMα1a (IMα1a), OsIMα1b (IMα1b), and their deletion mutants. NLS, nuclear localization signal; WD, WRKY domain; ARM, armadillo repeats; IBB, importin-β-binding domain; XB, exportin binding domain. b W76.1 and its deletion mutants were fused to the Gal4 DNA-binding domain (BD). IMα1a, IMα1b, IMαΔIBB1a, and IMαΔIBB1b were fused to the Gal4 activation domain (AD). Yeast cells with serial dilutions (10⁰, 10^–1, and 10^–2) were incubated in synthetic dropout medium lacking Leu and Trp (left panel) or Leu, Trp, His, and Ade (right panel) and photographed 3 days after plating. c Pull-down assays of OsIMαΔIBB1a and OsIMαΔIBB1b interacting with OsWRKY62 and OsWRKY76. All proteins were purified with their N-termini fused with GST and their C-termini fused with 3×flag or 3×myc tag. Each protein (about 1 µg) with its corresponding tag combination was incubated at 4 °C for 3 h in the immunoprecipitation (IP) buffer. The protein complexes were precipitated with anti-Flag affinity gel, washed five times with the IP buffer, and separated on 10% SDS-PAGE gels. The proteins were detected by western blots with anti-Myc and anti-Flag antibodies. Similar results were obtained from three repeats. NLS, nuclear localization signal; WD, WRKY domain

Fig. 2

OsIMα1a and OsIMα1b interacting with OsWRKY62.1 and OsWRKY76.1 primarily in the nuclei. a BiFC visualizations of OsWRKY62.1 (W62.1) and OsWRKY76.1 (W76.1) interacting with OsIMα1a and OsIMα1b. W62.1 and W76.1 were fused in frame with YFP N-terminal region (YFP^N) and IMαIBB1a and IMαIBB1b were fused with YFP C-terminal region (YFP^C). The plasmids indicated were introduced into N. benthamiana leaves through agroinfiltration method. Confocal images were taken 72 h after the treatments. Red fluorescence (dsRED^NLS) shows nuclear localization. From left panels to right: YFP images (YFP), dsRED images (RED), and combined YFP and RED in the bright field (Merged). b Co-localization of OsW62.1 with OsIMα1a and OsIMα1b. Plasmid 35S::W62.1-GFP or in combination with plasmid indicated was delivered into N. benthamiana leaves through agroinfiltration. Images were photographed 72 h after the treatments. DAPI (4',6-diamidino-2-phenylindole) for nuclear staining. From left panels to right: GFP, RED, DAPI, and the bright field image combined the fluorescent images (Merged). c Relative quantity of OsWRKY62.1-GFP localized in the nuclei. More than 100 cells from (b) were counted. Significant differences (Duncan's multiple range test; α = 0.05) compared with 35S::W62.1-GFP only are listed in the figure. Bar = 20 µm

Fig. 3

Interaction of OsIMαΔIBB1a and OsIMαΔIBB1b with the WD of OsWRKY62. a WD sequences of OsWRKY62 and OsWRKY76. The basic amino acids in red are mutated to Alanine. The amino acid differences between W62WD and W76WD are underlined in W76WD. b Pull-down assays of OsIMαΔIBB1a and OsIMαΔIBB1b interacting with W62WD and its mutants. The W62WD and its mutants were sandwiched with GST and 3myc tags and expressed. Each protein (about 1 µg) was incubated with GST-IMαΔIBB1a-3flag or GST-IMαΔIBB1b-3flag at 4 °C for 3 h in the IP buffer. The protein complexes were precipitated, washed five times with the IP buffer, separated on 10% SDS-PAGE gels, and detected by western blots with anti-Myc and anti-Flag antibodies. GST-3myc was used as a negative control. Similar results were obtained from three repeats. WD^AA, WD^AAA, and WD^5A for RK, KKK, and both of them in W62WD, respectively, were all mutated to Alanines. c The concatenated basic amino acids in W62WD were important for nuclear localization. W62WD and W62WD^5A were cloned in frame with GFP-GUS chimeric gene, respectively. The generated plasmid in combination with 35S::IMαΔIBB1a-dsRED were introduced into the leaf cells of N. benthamiana. Confocal images were taken 72 h after the agroinfiltration. From left panel to right: GFP images (GFP), dsRED images (RED), and combined GFP and RED in the bright field (Merged). d Interactions of IMαΔIBB1a with W62WD and AvrPib. W62WD, AvrPib, and their mutants were fused in frame with YFP N-terminal region (YFP^N), and IMαΔIBB1a was fused with YFP C-terminal region (YFP^C). The plasmids indicated were transformed into N. benthamiana leaves. Confocal images were taken at 72 h after the treatments. Red fluorescence (35S::dsRED^NLS) shows nuclear localization. From left panels to right: YFP images (YFP), dsRED images (RED), and combined YFP and RED in the bright field (Merged). Bar = 20 µm

Fig. 4

OsIMα1a and OsIMα1b were positively involved in resistance against rice blast pathogen. a The transgenic and wild-type (ZH17) plants of about 3-month-old were inoculated with a virulent strain of M. oryzae SZ by infiltration. The symptoms were photographed 10 days after the challenge. Bar = 2 cm. b Lesion length was measured from the inoculated leaves. P-values were calculated by one-tailed Student's t-test, n ≥ 8. The experiment was repeated twice with similar results. c–j Gene expression was determined by qRT-PCR analyses using rice ubiquitin gene as an internal standard. Values represent means ± SD (n = 3). Two independent experiments were performed and obtained similar results. CDU for overexpression of OsIMα1a and OsIMα1b, KO for knockout, and imα1abKO for double knockout mutants of OsIMα1a and OsIMα1b. Significant differences (Student's t-test) compared with the wild-type plants are listed in the figure (P-value < 0.05, *; < 0.01, **; < 0.001,***)

Fig. 5

OsIMα1a enhanced OsWRKY62.1 acting as a negative regulator of resistance against rice blast pathogen. a The 3-month-old transgenic and wild-type ZH17 plants were inoculated with a virulent strain of M. oryzae SZ by infiltration. Symptoms were photographed 10 days after the challenge. Bar = 2 cm. b Lesion length was measured from the inoculated leaves. Significant differences (Duncan's multiple range test; α = 0.05) compared with the wild-type plants are listed in the figure. c Change of OsWRKY62.1-GFP localization through mutation of NES-like sequence in OsWRKY62.1. The key amino acids in NES-like sequence of OsWRKY62.1 were mutated to A residues (W62.1^nes4A). The plasmids of 35S::W62.1^nes4A-GFP and 35S::W62.1-GFP, in combination with 35S::dsRED^NLS were introduced into the leaf cells of N. benthamiana, respectively. Confocal images were taken 72 h after the agroinfiltration. From left panel to right: GFP images (GFP), dsRED images (RED), and combined YFP and RED in the bright field (Merged). Bar = 20 µm