Actomyosin-driven motility and coalescence of phase-separated viral inclusion bodies are required for efficient replication of a plant rhabdovirus.

Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Fuels for ROS signaling in plant immunity.

Reactive oxygen species (ROS) signaling has an important role in plant innate immune responses and is primarily mediated by NADPH oxidase, also known as respiratory burst oxidase homologs (RBOHs) in plants. NADPH serves as a fuel for RBOHs and limits the rate or amount of ROS production. Molecular regulation of RBOHs has been extensively studied; however, the source of NADPH for RBOHs has received little attention. Here, we review ROS signaling and the regulation of RBOHs in the plant immune system with a focus on NADPH regulation to achieve ROS homeostasis. We propose an idea to regulate the levels of NADPH as part of a new strategy to control ROS signaling and the corresponding downstream defense responses.

Overexpression of an Antioxidant Enzyme APX1 in cpr5 Mutant Restores its Pleiotropic Growth Phenotype.

Breeding crops with enhanced immunity is an effective strategy to reduce yield loss caused by pathogens. The constitutive expresser of pathogenesis-related genes (cpr5) mutant shows enhanced pathogen resistance but retarded growth; thus, it restricts the application of cpr5 in breeding crops with disease resistance. Reactive oxygen species (ROS) play important roles in plant growth and defense. In this study, we determined that the cpr5 mutant exhibited excessive ROS accumulation. However, the mutation of respiratory burst oxidase homolog D (RBOHD), a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for the production of ROS signaling in plant immunity, did not suppress excessive ROS levels in cpr5. Furthermore, the cpr5 mutant showed low levels of ascorbate peroxidase 1 (APX1), an important cytosolic ROS-scavenging enzyme. APX1 overexpression in the cpr5 background removed excessive ROS and restored the pleiotropic growth phenotype. Notably, APX1 overexpression did not reduce the resistance of cpr5 mutant to virulent strain Pseudomonas syringae pv. tomato (Pst) DC3000 and avirulent strain Pst DC3000 (avrRpt2). These results suggest that the removal of excessive ROS by APX1 overexpression restored the cpr5 growth phenotype while conserving pathogen resistance. Hence, our study provides a theoretical and empirical basis for utilizing CPR5 in the breeding of crops with disease resistance by effective oxidative stress management via APX1 expression.

The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production.

Reactive oxygen species (ROS) production is a conserved immune response in Arabidopsis primarily mediated by respiratory burst oxidase homolog D (RBOHD), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase associated with the plasma membrane. A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production. However, the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear. In this study, we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4 (FIN4), which encodes the first enzyme in de novo NAD biosynthesis. fin4 mutants show reduced NADPH levels and impaired ROS production. However, FIN4 and other genes involved in NAD- and NADPH-generating pathways are not highly upregulated upon elicitor treatment, raising a possibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD. To verify this possibility, we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE (RIPK), a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity, and identified NADP-malic enzyme 2 (NADP-ME2), an NADPH-generating enzyme. Compared with wild-type plants, nadp-me2 mutants display decreased NADP-ME activity, lower NADPH levels, and reduced ROS production in response to immune elicitors. Furthermore, we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro. The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum. Collectively, our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH, thus fueling RBOHD to sustain ROS production in plant immunity.

RIPK1 inhibitor ameliorates the MPP+/MPTP-induced Parkinson's disease through the ASK1/JNK signalling pathway.

Receptor-interacting protein kinase 1 (RIPK1) is up-regulated in patients with neurodegenerative diseases. Our study aimed to explore the underlying mechanisms that involved in the neurotoxic function of RIPK1 in Parkinson's disease (PD). MPP+/MPTP-induced PD cellular and mice models were used in this study. The results showed that RIPK1 was high expressed and activated in MPP+-treated SH-SY5Y cells and MPTP-induced PD mice. Overexpression of RIPK1 facilitated cell apoptosis, necrosis, inflammation response, ROS production and mitochondrial dysfunction in MPP+- treated SH-SY5Y cells, while the RIPK1 inhibitor Nec-1s has an opposite effect. In addition, the Apoptosis-signaling kinase-1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated during the overexpression of RIPK1, and inhibiting the ASK1/JNK signal by the ASK1 inhibitor partially reversed the decline of cell viability, the increase of cell apoptosis, necrosis and inflammation induced by RIPK1 overexpression in MPP+-treated SH-SY5Y cells. Further studies suggested that the inhibition of RIPK1 by Nec-1s largely alleviated the behavioural impairment in PD mice. Hence, our study indicated that the RIPK1 inhibitor Nec-1s has neuroprotective effects against PD through inactivating the ASK1/JNK signalling pathway.

Validation of reference genes for RT-qPCR analysis in Burkholderia pyrrocinia JK-SH007.

Burkholderia pyrrocinia strain JK-SH007 isolated from poplar stems plays a highly significant role in the growth promotion and the biocontrol of poplar canker during colonization in poplar. In this research, the ideal reference gene was filtered and determined for the transcript normalization. Additionally, the expression of pyrG under all four conditions was relatively stable in B. pyrrocinia JK-SH007.