Volume 11, Issue 2, Pages 338-341 (February 2018) Barley Stripe Mosaic Virus γb Interacts with Glycolate Oxidase and Inhibits Peroxisomal ROS Production to Facilitate Virus Infection  Meng Yang, Zhenggang Li, Kun Zhang, Xuan Zhang, Yongliang Zhang, Xianbing Wang, Chenggui Han, Jialin Yu, Kai Xu, Dawei Li  Molecular Plant  Volume 11, Issue 2, Pages 338-341 (February 2018) DOI: 10.1016/j.molp.2017.10.007 Copyright © 2017 The Author Terms and Conditions

Figure 1 Facilitation of BSMV Infection by γb-GOX Interactions and Inhibition of ROS Bursts. (A) Analysis of γb repression of H2O2 and O2− bursts in N. benthamiana leaves by NBT or DAB staining for O2− or H2O2 levels at 3 days after agroinfiltration. Empty vector (EV) negative control, BSMV infection, γb transient expression (γb), and BSMV γb deletion mutant (BSMVΔγb) infection. NBT and DAB staining intensities are quantified in Supplemental Figure 2. (B) Co-immunoprecipitation assays for analysis of interactions between γb and GOX. The GOX-HA protein and γb-3×Flag or GUS-3×Flag proteins were co-expressed by agroinfiltration of N. benthamiana leaves. GUS-3×Flag and GOX-HA co-expression served as the negative controls. Upper panels, anti-HA antibody detection; lower panels, anti-Flag antibody detection. H chain and L chain positions are identified along the right side of the panels. White triangles indicate precipitated proteins using anti-Flag beads. (C) Depiction of GOX enzymatic activity at 3 days after agroinfiltration: γb transient expression, BSMV or BSMVΔγb infection, RNAα + RNAγ (movement deficient), or RNAα + RNAγΔγb infiltrations. Error bars indicate the SD from the mean of three biological replicates, asterisks represent significant differences (t-test, ∗p < 0.05; ∗∗∗p < 0.001). (D) Peroxisomal redox status of GOX overexpressing, GOX + γb co-overexpressing, γb transgenic (γb transgene) or non-transgenic (NT) plant leaves 3 days after agroinfiltration of roGFP2-SKL (Supplemental Figure 14). Ratiometric images and average ratio values of fluorescence excited by 405 nm and 488 nm lasers are shown in each panel. Color scale of the ratio values is shown on the right: white color indicates most oxidized state and blue color indicates most reduced state. Scale bars represent 100 μm. (E) DAB staining analysis of H2O2 bursts in BSMV-infected plants in which gox or gus controls were silenced by TRV-gox or TRV-gus. Relative DAB staining intensity of H2O2 levels are shown in Supplemental Figure 8. (F) Inhibition of BSMV infection after GOX silencing. TRV-gus VIGS control; TRV-gox VIGS. Representative image of gox-silenced (or gus-silenced) leaves infiltrated with BSMVγb-GFP at 3 dpi (left). GFP fluorescence was excited at 488 nm. Scale bars represent 100 nm. GOX mRNA (upper right) or GOX protein levels (lower right) in gox-silenced or control plants were measured via real-time PCR and western blotting, respectively. (G) Effects of GOX overexpression on BSMV infection. BSMVγb-GFP was agroinoculated into plant leaves agroinfiltrated with EV, GOXm, or GOX. The BSMVγb-GFP infection foci were photographed under confocal laser microscopy. Scale bars represent 60 μm. (H) Northern blots detecting BSMV RNA accumulation with a radioactive probe complementary to the 3′-UTR of BSMV RNAs (upper two panels). GOX protein levels were analyzed by western blotting (lower two panels). Molecular Plant 2018 11, 338-341DOI: (10.1016/j.molp.2017.10.007) Copyright © 2017 The Author Terms and Conditions