GoKawiila, The candidate XA48-interacting proteins revealed by Y2H screen. Note that OsVOZ1 is the most hit protein. b, Western blots detected expression of XA48-CC-nLuc, cLuc-OsVOZ1 and cLuc-OsVOZ2 in N. benthamiana for protein interaction. c, Representative images of XA48 subcellular localization. XA48-YFP was transiently expressed in rice protoplasts, which showed main localization to the cell periphery and nucleus. OsRAC1-mCherry and NLS-mCherry served as a plasma membrane (PM) and nuclear marker, respectively. Scale bars, 10 µm. d, Representative images showing the subcellular localization of OsVOZ1 and OsVOZ2 at the cell periphery and nucleus. OsVOZ1-YFP and OsVOZ2-YFP were transiently expressed in rice protoplasts. Scale bars, 10 µm. e, f, Co-localization of XA48 with OsVOZ1 and OsVOZ2 was detected in cell periphery and nucleus (e), and the presence of XopG did not alter the co-localization pattern of XA48 with OsVOZ1/OsVOZ2 (f). OsVOZ1-mCherry, OsVOZ2-mCherry, Xa48-CFP and XopG-GFP were transiently expressed in rice protoplasts. Scale bars, 10 µm. g-h, qRT-PCR and immunodetection analyses were performed on OsVOZ1-GFP and OsVOZ2-GFP transgenic overexpression (OE) lines, using SKZ and NIPB-Xa48 as wild type controls. Actin was detected as a loading control (mean ± SD, two-tailed t test; n = 3). i, Schematic of OsVOZ1/2 knockout (KO) lines in NIPB background. j, The degradation of OsVOZ1 and OsVOZ2 by XopG and XA48-CC in vitro was inhibited by the zinc metalloprotease-specific inhibitor phenanthroline. Purified OsVOZ1-MBP, OsVOZ2-MBP, XopG-MBP and XA48-CC-His were incubated in reaction mixture with or without 5 mM phenanthroline. Protein levels were analysed by Coomassie blue staining and Western blotting. k, XA48-CC promoted the interaction between XopG and OsVOZs. Notably, in vitro pull-down assays showed that OsVOZ1-MBP and OsVOZ2-MBP pulled down XopG-His in the presence of XA48-CC-GST. l, Resin sections of leaves from NIPB, CR-Osvoz1, and CR-Osvoz2 mutants. Microscopic observation revealed that the CR-Osvoz1 and CR-Osvoz2 mutants developed abnormal vascular bundles, including reduced xylem and phloem size, impaired xylem cell differentiation, and irregular vessel morphology. Xylem (xy), phloem (ph), and vessels (ve) are highlighted with in red, yellow, and black lines, respectively. m, The areas of xylem and phloem were measured, revealing that their sizes in the CR-Osvoz1 and CR-Osvoz2 mutants were smaller than those in NIPB (mean ± SD, two-tailed t test; n = 8). Experiments were independently repeated twice with similar results (b-f, j, k).
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