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

Abstract

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOHderived ROS was demonstrated for the replication of another (+) RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.positive-strand RNA virus | viral RNA replication | reactive oxygen species | respiratory burst oxidase homolog | calcium-dependent protein kinase P lants have evolved complicated and sophisticated strategies to survive environmental changes. The rapid generation of reactive oxygen species (ROS) is one of the hallmarks of plant responses to various biotic and abiotic stresses (1). 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 (5-8). RBOH activity is tightly and coordinately regulated posttranslationally [e.g., activation by Ca 2+ , phosphatidic acid (PA), G proteins, or protein kinases] (9-12). It has recently been shown that receptor-like cytoplasmic kinases and calciumdependent protein kinases (CDPKs) regulate RBOHs activity via direct phosphorylation or through modulating regulators of RBOHs during recognition of MAMPs via corresponding surface-loca...

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus

“…S7a). p88 pol also interacts with NbRbohB (Hyodo et al ., ). However, NbRACK1 silencing had no impact on the p88 pol –NbRbohB interaction (Fig.…”

“…We previously proposed that RCNMV infection or its replication protein p27 triggers intracellular ROS production in the cytoplasm via hijacking the NbRbohB–NbCDPKiso2 complex (Hyodo et al ., ). NbCDPKiso2 phosphorylates and activates NbRbohB and plays an important role in p27‐induction of ROS accumulation (Hyodo et al ., ). The observation that NbRACK1 associates with both viral replication protein p27 and NbCDPKiso2 (Figs ) and mediates bridging between p27 and NbCDPKiso2 (Fig.…”

“…These include heat shock protein 70 (Hsp70), Hsp90, small GTPase ADP‐ribosylation factor 1, a phosphatidic‐acid‐generating enzyme phospholipase D, reactive oxygen species (ROS)‐generating enzyme respiratory burst oxidase homologue B (RbohB), and calcium‐dependent protein kinase isoform 2 (CDPKiso2) (Mine et al ., ; Hyodo et al ., , , ). RCNMV triggers cytoplasmic ROS accumulation via the replication protein p27 for robust viral RNA replication in infected cells, and p27‐mediated induction of cytoplasmic ROS accumulation depends on RbohB and CDPKiso2 (Hyodo et al ., ). Among the ROS, O 2 − appears to be required for RCNMV replication (Hyodo et al ., ).…”

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

“…To confirm the effect of OsRboh ‐mediated ROS accumulation on RBSDV infection, diphenyliodide (DPI)‐treated rice plants were inoculated with RBSDV. DPI is a well‐known inhibitor of RBOH activity (Hyodo et al , ; Lv et al , ). As shown in Fig.…”

“…However, the interplay between auxin and ROS has been little reported in the context of plant defence. Recently, ROS were demonstrated to be involved in plant–virus interactions (Hyodo et al , ; Wu et al , ; Xie et al , ). Two positive‐strand RNA viruses, Red clover necrotic mosaic virus (RCNMV) and Brome mosaic virus , were shown to hijack the host's ROS‐generating machinery and trigger intracellular ROS bursts (Hyodo et al , ).…”