Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Abstract

These authors contributed equally to this work. SUMMARYBrassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H 2 O 2 and NO. Scavenging of H 2 O 2 or NO in upper leaves blocked BR-induced systemic virus resistance. BR-induced systemic H 2 O 2 accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite-dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR-activated H 2 O 2 is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H 2 O 2 generation blocked BR-induced systemic NO production, but BR-induced H 2 O 2 production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR-induced systemic virus defense in NbRBOHB-silenced plants, but H 2 O 2 did not reverse the effect of NbNR silencing on BR-induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR-mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR-induced H 2 O 2 and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR-mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)-dependent H 2 O 2 production and subsequent systemic NR-dependent NO generation.

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

“…NbRBOHB-derived ROS play a positive role in plant immunity against several plant pathogens including fungi, bacteria, and viruses (5)(6)(7)(8). Therefore, we first expected that RCNMV replication proteins would be recognized by RBOHs to trigger ROS production to inhibit viral infection, or that RCNMV replication proteins act as a suppressor of RBOHs to counteract RBOHmediated antiviral immunity.…”

“…During pathogen infection plants rapidly produce ROS to induce local or systemic signaling through the activation of cell surface-localized RBOH proteins. ROS signaling mediates systemic resistance against plant viruses (7,8) and is therefore considered to be a positive regulator of plant antiviral defense. However, the precise function of ROS in plant-virus interaction has not been extensively studied.…”

“…Plant NADPH oxidase, termed RBOHs (respiratory burst oxidase homologs), localize at the plasma membrane (PM) and intracellular compartments including the Golgi apparatus (2,3) and play a key role in ROS production upon the perception of environmental stresses (4). In Nicotiana benthamiana, an RBOHB (NbRBOHB) plays important roles in ROS production triggered by microbe-associated molecular patterns (MAMPs), such as bacterial flagellin and fungal chitin, and facilitates plant immunity against biotrophic pathogens including an oomycete pathogen Phytophthola infestance and tobacco mosaic virus (5)(6)(7)(8). RBOH activity is tightly and coordinately regulated posttranslationally [e.g., activation by Ca 2+ , phosphatidic acid (PA), G proteins, or protein kinases] (9-12).…”

The Roles of Alpha-Momorcharin and Jasmonic Acid in Modulating the Response of Momordica charantia to Cucumber Mosaic Virus

“…CMV was maintained in an aqueous suspension of 0.02 M sodium phosphate buffer (PBS, pH 7.0) at 4°C. The inoculation with virus was implemented as described previously (Deng et al, 2016b). PBS buffer was used as a control.…”

“…M. charantia leaves were vacuum infiltrated with NBT (0.5 mg/mL) solutions for 2 h. Leaves were then decolorized in boiling ethanol (90%) for 15 min. The method used for H 2 O 2 fluorescence probe staining was described by Deng et al (2016b). The Amplex red hydrogen peroxide/peroxidase assay kit (Invitrogen, Waltham, MA, USA) was used to determine H 2 O 2 accumulation.…”