Journal: bioRxiv

Article Title: The mitogen-activated protein kinase kinase kinase, ILK5, regulates plant purinergic receptor-mediated, innate immunity

doi: 10.1101/2022.04.19.488815

Figure Lengend Snippet: ( A ) Schematic diagram of ILK5 (At4g18950) showing the ankyrin repeat (ANK) and serine-threonine or tyrosine kinase (S-T/Y kinase) domains. Identified phosphopeptide VKKLDDEVLS(p) is located in the kinase domain. ( B, C , F, G ) LCI experiment showing interaction of P2K1-ILK5 protein with/without 200 μM ATP treatment. The luminescence was monitored and captured using a low light imaging CCD camera (Photek; Photek, Ltd.). Dotted circles indicate the infiltrated area in N. benthamiana leaves. MKK3 protein was used as a negative control. ( D ) BiFC assay in Arabidopsis protoplasts. The YFP fluorescence was monitored using a Leica DM 5500B Compound Microscope with Leica DFC290 Color Digital Camera 24 h after transformation. DAPI and FM464 were used as a nuclear marker and plasma membrane marker, respectively. Chl represents chlorophyll auto-fluorescence signal. Merge indicates overlapped image of YFP and FM4-64. RBOHD and ILK6 were used as positive and negative control, respectively. Enlarged indicates enlargement of YFP image to clearly indicate plasma membrane-associated yellow fluorescence. Scale bars: 10 μm. ( E ) Co-immunoprecipitation of P2K1 and ILK5 proteins. The indicated constructs were co-infiltrated and transiently expressed in N. benthamiana leaves after addition of 200 μM ATP (+) for 30 min or MES buffer (pH 5.7) as a mock treatment (−). Total protein was used for Co-IP. Anti-HA and anti-Myc antibodies were used. MKK8-Myc was used as a negative control. ( H, I ) P2K1 directly phosphorylates ILK5 at Ser192. Purified recombinant ILK5-His and ILK5 S192A protein was incubated with GST-P2K1 kinase domain (GST-P2K1-KD), P2K1 kinase dead versions, (GST-P2K1-KD-1; D572N or GST-P2K1-KD-2; D525N), or GST in an in vitro kinase assay. Auto- and trans-phosphorylation were detected by incorporation of γ-[ 32 P]-ATP. MBP and CPK5 were used as a universal substrate and a negative control, respectively. Protein loading was visualized by coomassie brilliant blue (CBB) staining. ( J ) Quantification of phosphorylated ILK5 and ILK5 S192A protein. The intensity of the phosphorylation signals of ILK5 and ILK5 S192A by P2K1-KD (shown in SI Appendix, ) were measured and analyzed using the Image J and GraphPad Prism 8. Data shown as mean ± SD, n = 4, ** p <0.01, unpaired Student’s t test, two-way. ( K ) ILK5 protein is phosphorylated by ATP treatment in vivo . CPK5-HA used as a negative control. ( L ) Mutation of ILK5 at Ser192 results reduced phosphorylation under ATPγS treatment. ( M ) ILK5 phosphorylation is dependent on P2K1 protein. In panel K - M , either ILK5-HA or ILK5 S192A -HA protein was expressed in Arabidopsis WT, p2k1-3 or P2K1-OX protoplasts after addition of 250 μM ATPγS and subsequently immunoprecipitated (IP) using anti-HA antibody beads and immunoblotting (IB) was carried out with an anti-phospho-Ser/Thr antibody. Protein loading was visualized by CBB staining. Above experiments were repeated at least two times with similar results.

Article Snippet: The luminescence was monitored and captured using a low light imaging CCD camera (Photek; Photek, Ltd.).

Techniques: Imaging, Negative Control, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy, Transformation Assay, Marker, Immunoprecipitation, Construct, Co-Immunoprecipitation Assay, Purification, Recombinant, Incubation, In Vitro, Kinase Assay, Staining, In Vivo, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: The mitogen-activated protein kinase kinase kinase, ILK5, regulates plant purinergic receptor-mediated, innate immunity

doi: 10.1101/2022.04.19.488815

Figure Lengend Snippet: ( A, H ) Three-week-old plants were flood inoculated with luxCDABE -tagged Pst DC3000 suspension (OD 600 = 0.002 approximately 5 × 10 6 CFU mL −1 ) containing 0.025% (v/v) Silwet L-77 in the presence of ATP (250 μM). Inoculated plants [either ilk5-1 mutants, complemented ilk5-1 mutant with ILK5 WT -HA (L1), or ILK5 S192A -HA mutant (L1) plants] were used and luciferase luminescence was detected using a low light capture CCD camera either at the time of inoculation (0 days) and 3 days post inoculation. sid2-2 was used as a control. ( B , I ) Quantification of luminescence signal intensities. The signal of luxCDABE -tagged Pst DC3000 for each plant was monitored, images were captured, and the luciferase signal intensities were quantified using the C-vision/Im32. ( C, J ) Bacterial colonization was determined by plate counting. In panel B (n = 9), C (n = 8), I (n = 6), J (n = 8), data shown as mean ± SEM, **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, p -value indicates significance relative to wild-type plants and was determined and analyzed using the GraphPad Prism 8 by one-way ANOVA followed by Dunnett’s multiple comparisons. ( D , E ) Real-time qRT-PCR analysis of WRKY40 and CPK28 transcripts in p2k1-3, ilk5-1 and ilk5-3 mutant backgrounds after ATP (250 μM) treatment. Total RNA was isolated from 2-week-old plants and 2 μg of total RNA was used in this experiment. The SAND reference gene was used for data normalization. Data shown as mean ± SD, WRKY40; n = 4, CPK28; n = 6, **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, p -value was determined and analyzed using the GraphPad Prism 8 by unpaired two-way Student’s t test. These experiments were repeated two times (biological replicates) with similar results. ( F , G, K ) Phosphorylation of MPK3/MPK6 detected in ilk5 mutants or complemented lines in which ILK5 or ILK5 S192A expression was driven by the native promoter by western blotting using anti-phospho 44/42 antibody. ATPγS (250 μM) was added and plants incubated for the times shown. Total protein was extracted at each time point and IB was performed with an anti-44/42 MAPK antibody. CBB staining of protein was used as a loading control. ( L ) Hypothetical model for the role of ILK5 in eATP signaling. Upon addition of the activating ligand eATP, the P2K1 receptor is rapidly auto-phosphorylated and then directly interacts and phosphorylates its downstream target, the ILK5 protein, leading to innate immune response via MAPK cascades. Above the experiments were repeated at least two times (biological replicates) with similar results.

Article Snippet: The luminescence was monitored and captured using a low light imaging CCD camera (Photek; Photek, Ltd.).

Techniques: Mutagenesis, Luciferase, Quantitative RT-PCR, Isolation, Expressing, Western Blot, Incubation, Staining