Journal: Nature Plants

Article Title: De-repression of protein phosphatase 5 by the chaperone organizer HOP1 activates plant NLR immunity

doi: 10.1038/s41477-026-02253-4

Figure Lengend Snippet: a , LET7 PPase does not interact with TPR5G. Recombinant HIS-LET7 PPase proteins purified from E. coli were immobilized on Ni-NTA biosensor chips and incubated with soluble GST-LET7 TPR , GST-TPR5G, or GST proteins as analyte. The buffer served as a control (Ctrl). The graph shows the association (0 to 180 sec) and dissociation (180 to 360 sec) times of the interaction (left). CBB staining shows the recombinant protein expression (right). b , AlphaFold-based prediction of LET7 and its crystal structure. Structural models generated with AlphaFold 2 of full-length LET7 (left panel). The AlphaFold-predicted structure of LET7 is reminiscent of its crystal structure with the intramolecular interaction between LET7 TPR and LET7 PPase . X-ray crystal structure of LET7 showing autoinhibition of the catalytic LET7 PPase domain (green) by binding to the N-terminal TPR domain (blue) (PDB code: 7OBE) (right panel). LET7 structure figures were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 2.0, DeLano Scientific, Palo Alto, CA, 1998, https://pymol.org/2/ ). c , Potential LET7-interacting proteins identified by Y2H screens. LET7 in the pGBKT7 vector was transformed into the yeast AH109 strain. The resulting yeast transformants were then transformed with the Arabidopsis cDNA library in the pGADT7 vector, followed by screening using SD-HLT medium supplemented with 1 mM 3-AT. In-frame candidates with the number of independent colonies (count) carrying the corresponding genes are shown. d , Genotyping PCR of three hop1 mutant alleles. Genomic DNAs from WT, hop1-1 ( sail_734_f01 ), hop1-2 ( gabi_420a10 ), and hop1-3 ( salk_052232 ) were used for PCR analysis to check the annotated T-DNA insertions. The primer pair of LP and RP is used to amplify the genomic DNA fragment of HOP1 , and the primer pair of LB and RP is used to amplify the T-DNA insertions. WT seedlings were used as a negative control. e , Genotyping PCR confirms the mekk1 hop1-2 double mutant. Genomic DNAs from the F 2 generation seedlings of the heterozygous mekk1 + /- mutant crossed with hop1-2 were screened by PCR. The primer pair of LP and RP amplifies the genomic DNA fragment of HOP1 or MEKK1 , and the primer pair of LB and RP amplifies the T-DNA insertions. WT seedlings were used as a negative control. f , The rar1 mutant does not obviously suppress RNAi- MEKK1 or RNAi- BAK1/SERK4- mediated cell death. VIGS assays were performed as in Fig. using rar1-21 . Photographs were taken three weeks after inoculation. Scale bar, 1 cm. g , HOP1 TPR1 interacts with LET7 TPR in Y2H assays. Full-length or truncated versions of HOP1 and LET7 were cloned into pGBKT7 and pGADT7 vectors for Y2H assays. Experiments were performed similarly to those in Fig. . Experiments ( a , d-g ) were repeated three times with similar results.

Article Snippet: Genes cloned in recombinant protein expression vectors were induced and expressed in E. coli BL21 strain (DE3) at 16 °C using LB medium supplemented with 0.4 mM isopropyl-β- D -1-thiogalactopyranoside for 12–18 h. HIS fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen), and GST fusion proteins were purified with Pierce glutathione agarose (Thermo Scientific) according to the standard protocols provided by the manufacturers.

Techniques: Recombinant, Purification, Incubation, Control, Staining, Expressing, Generated, Binding Assay, Plasmid Preparation, Transformation Assay, cDNA Library Assay, Mutagenesis, Negative Control, Clone Assay

Journal: Nature Plants

Article Title: De-repression of protein phosphatase 5 by the chaperone organizer HOP1 activates plant NLR immunity

doi: 10.1038/s41477-026-02253-4

Figure Lengend Snippet: a , Schematic diagram of mutant lines and protein motifs of HOP1. Top: T-DNA insertions in HOP1 (AT1G12270) for three mutant lines are shown with three T-DNA insertional lines. The open boxes are 5′ and 3′ UTRs, the grey boxes indicate exon protein-coding regions and the lines indicate introns. Bottom: protein motifs, including TPR1, TPR2a and TPR2b, with amino acid positions, are labelled. b – d , The hop1 mutants suppress RNAi -MEKK1 -triggered growth defects, cell death, H 2 O 2 production and PR1 gene expression. WT and hop1 plants are shown three weeks after inoculation with Agrobacterium carrying the VIGS vector targeting GFP as a control (Ctrl) or RNAi -MEKK1 ( b ). Scale bars, 1 cm. Detached leaves were stained with trypan blue for cell death or DAB for H 2 O 2 accumulation ( c ). Scale bars, 0.5 cm. The expression of PR1 was normalized to that of ACTIN2 , and the data are shown as means ± s.d. ( n = 3, biologically independent samples) ( d ). Column 1 versus Column 2, P < 0.0001, Column 1 versus Column 3, P > 0.9999, Column 3 versus Column 4, P = 0.0222. e , f , The hop1-2 mutant suppresses growth defects, cell death and the elevated expression of PR1 and PR2 in mekk1 . Three-week-old soil-grown plants are shown ( e , top) with leaves stained by trypan blue for cell death ( e , bottom). Scale bars, 1 cm. The expression of PR1 and PR2 was normalized to ACTIN2 . PR1 : Column 1 versus Column 3, P < 0.0001, Column 3 versus Column 4, P < 0.0001, Column 2 versus Column 4, P = 0.0003; PR2 : Column 1 versus Column 3, P < 0.0001, Column 3 versus Column 4, P < 0.0001, Column 2 versus Column 4, P = 0.0012. g , h , Expression of HOP1–GFP restores RNAi- MEKK1 -induced growth defects and PR1 expression in hop1-2 . HOP1 tagged with GFP under the 35S promoter ( p35S :: HOP1-GFP ) was transformed into hop1-2 . Scale bars, 1 cm ( g , top). Immunoblotting by an anti-GFP antibody shows HOP1–GFP proteins with CBB staining RBC as a loading control ( g , bottom). The expression of PR1 was normalized to ACTIN2 ( h ). Column 1 versus Column 2, P > 0.9999, Column 1 versus Column 3, P > 0.9999, Column 1 versus Column 5, P < 0.0001, Column 2 versus Column 3, P > 0.9999, Column 2 versus Column 4, P > 0.9999, Column 5 versus Column 6, P < 0.0001, Column 6 versus Column 7, P < 0.0001, Column 6 versus Column 8, P < 0.0001. i , LET7 interacts with HOP1 in Y2H assays. The experiments were performed similarly to those in Fig. . j , LET7 associates with HOP1 in BiFC assays. LET7 or HOP1 was fused with the N-terminal or C-terminal half of YFP (LET7–nYFP, LET7–cYFP, HOP1–cYFP or HOP1–nYFP) and co-expressed in N. benthamiana leaves with empty vectors carrying nYFP or cYFP (EV–nYFP or EV–cYFP) as controls. Signals were observed via confocal microscopy at 48 h post-inoculation. Scale bars, 25 μm. k , LET7 associates with HOP1 in Co-IP assays. LET7–HA was co-expressed with HOP1–GFP or GFP in protoplasts. Total proteins were immunoprecipitated with anti-HA affinity beads, followed by immunoblotting with an anti-GFP or anti-HA antibody (top two panels). Proteins before immunoprecipitation were immunoblotted and are shown as input controls (bottom two panels). l , LET7 interacts with HOP1 in pull-down assays. GST or GST–HOP1 immobilized on glutathione agarose was incubated with HIS–LET7 proteins. Top: washed beads were pelleted for immunoblotting using an anti-HIS antibody. Middle and bottom: input proteins are shown with immunoblotting before pull-down. m , LET7 interacts with HOP1 in BLI assays. Recombinant HIS–LET7 proteins were immobilized on Ni-NTA biosensor chips and incubated over a range of concentrations (1.25–10 μM) of soluble GST–HOP1 proteins as the analyte. The plot shows the association (0 to 180 s) and dissociation (180 to 360 s) times of the interaction. The equilibrium K d of the LET7 and HOP1 interaction is 2.062 ± 0.031 μM as determined by Octet BLI analysis software. The data are shown as mean ± s.d. ( n = 3 biologically independent samples in d , f and h ). Different letters indicate significant differences determined by one-way analysis of variance followed by Tukey’s test ( P < 0.05). The experiments were repeated four times in b , e , i , l and m and three times in c , d , f – h , j and k with similar results.

Article Snippet: Genes cloned in recombinant protein expression vectors were induced and expressed in E. coli BL21 strain (DE3) at 16 °C using LB medium supplemented with 0.4 mM isopropyl-β- D -1-thiogalactopyranoside for 12–18 h. HIS fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen), and GST fusion proteins were purified with Pierce glutathione agarose (Thermo Scientific) according to the standard protocols provided by the manufacturers.

Techniques: Mutagenesis, Gene Expression, Plasmid Preparation, Control, Staining, Expressing, Transformation Assay, Western Blot, Confocal Microscopy, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, Recombinant, Software