The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense ...pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.
SYNOPSIS
The Barley stripe mosaic virus (BSMV) γb protein promotes viral infection in plants via various mechanisms. This work reveals an additional role in disrupting chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.
BSMV infection disturbs chloroplast redox homeostasis and induces oxidative stress.
The BSMV γb protein directly interacts with host NADPH‐dependent thioredoxin reductase C (NTRC) at chloroplasts.
NTRC expression suppresses BSMV replication in Nicotiana benthamiana.
γb protein competitively interferes with NTRC binding to 2‐Cys peroxiredoxins.
Inhibition of the chloroplast‐localized NADPH‐dependent thioredoxin reductase C to create an oxidative microenvironment promotes plant infection by a chloroplast‐replicating virus.
Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots ...and insect pests is challenging.
Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown plan-thopper (SBPH, Laodelphax striatellus) insect vector.
We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid β-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves.
Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.
Autophagy is a conserved defense strategy against viral infection. However, little is known about the counterdefense strategies of plant viruses involving interference with autophagy. Here, we show ...that γb protein from Barley stripe mosaic virus (BSMV), a positive single-stranded RNA virus, directly interacts with AUTOPHAGY PROTEIN7 (ATG7). BSMV infection suppresses autophagy, and overexpression of γb protein is sufficient to inhibit autophagy. Furthermore, silencing of autophagy-related gene ATG5 and ATG7 in Nicotiana benthamiana plants enhanced BSMV accumulation and viral symptoms, indicating that autophagy plays an antiviral role in BSMV infection. Molecular analyses indicated that γb interferes with the interaction of ATG7 with ATG8 in a competitive manner, whereas a single point mutation in γb, Tyr29Ala (Y29A), made this protein deficient in the interaction with ATG7, which was correlated with the abolishment of autophagy inhibition. Consistently, the mutant BSMVY29A virus showed reduced symptom severity and viral accumulation. Taken together, our findings reveal that BSMV γb protein subverts autophagy-mediated antiviral defense by disrupting the ATG7-ATG8 interaction to promote plant RNA virus infection, and they provide evidence that ATG7 is a target of pathogen effectors that functions in the ongoing arms race of plant defense and viral counterdefense.
Selective autophagy mediates specific degradation of unwanted cytoplasmic components to maintain cellular homeostasis. The suppressor of gene silencing 3 (SGS3) and RNA‐dependent RNA polymerase 6 ...(RDR6)‐formed bodies (SGS3/RDR6 bodies) are essential for siRNA amplification in planta. However, whether autophagy receptors regulate selective turnover of SGS3/RDR6 bodies is unknown. By analyzing the transcriptomic response to virus infection in Arabidopsis, we identified a virus‐induced small peptide 1 (VISP1) composed of 71 amino acids, which harbor a ubiquitin‐interacting motif that mediates interaction with autophagy‐related protein 8. Overexpression of VISP1 induced selective autophagy and compromised antiviral immunity by inhibiting SGS3/RDR6‐dependent viral siRNA amplification, whereas visp1 mutants exhibited opposite effects. Biochemistry assays demonstrate that VISP1 interacted with SGS3 and mediated autophagic degradation of SGS3/RDR6 bodies. Further analyses revealed that overexpression of VISP1, mimicking the sgs3 mutant, impaired biogenesis of endogenous trans‐acting siRNAs and up‐regulated their targets. Collectively, we propose that VISP1 is a small peptide receptor functioning in the crosstalk between selective autophagy and RNA silencing.
Synopsis
Genomic analyses have predicted numerous potentially expressed small peptides in plants, but their functions remain largely unknown. Here, a virus‐induced peptide is identified as a new autophagy receptor triggering degradation of SGS3/RDR6‐bodies and negatively regulating siRNA amplification.
VISP1 is a novel virus‐induced small peptide composed of 71 amino acids in Arabidopsis.
VISP1 harbors a ubiquitin‐interacting motif (UIM), which mediates the interaction with ATG8.
VISP1 serves as a new autophagy receptor and triggers autophagic degradation of SGS3/RDR6‐bodies.
VISP1 negatively regulates SGS3/RDR6‐dependent siRNA amplification.
VISP1, a small virus‐induced peptide, functions as an autophagy receptor to negatively regulate SGS3/RDR6‐dependent siRNA amplification in plants.
Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously ...demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys‐10, Cys‐19, and Cys‐60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non‐palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication‐movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell‐to‐cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.
Synopsis
Viral replication and subsequent intercellular movement are two critical steps for efficient plant infection. This work reveals induction of a replication‐to‐movement switch by palmitoylation of the viral γb protein during Barley stripe mosaic virus (BSMV) infection.
Non‐palmitoylated γb is anchored to the chloroplasts and promotes BSMV replication.
During the late stages of replication, S‐acyltransferases NbPAT15 and NbPAT21 palmitoylate γb at the periphery of chloroplasts.
Palmitoylated γb recruits the TGB1 movement protein to chloroplasts, thereby inducing a shift between viral replication and movement phases.
Palmitoylated γb promotes viral cell‐to‐cell movement by inhibiting NbREM1‐dependent callose deposition at plasmodesmata.
The transition from plant hordeivirus replication at the chloroplasts to its intercellular movement is induced by palmitoylation of the multifunctional viral γb protein.
Some plant and animal pathogens can manipulate their hosts to cause them to release odors that are attractive to the pathogens＇ arthropod vectors. However, the molecular mechanism underlying this ...process is largely unexplored, and the specific effectors the pathogens employ as well as the pathways within the hosts they target are currently unknown. Here we reveal that the aphid-borne cucumber mosaic virus （CMV） employs its 2b protein, a well-charac- terized viral suppressor of host RNA interference （VSR）, to target the host＇s jasmonate （JA） hormone pathway, thus acting as a viral inducer of host attractiveness to insect vectors （VIA）. 2b inhibits JA signaling by directly interacting with and repressing JA-induced degradation of host jasmonate ZIM-domain proteins, instead of using its VSR activ- ity. Our findings identify a previously defined VSR protein as a VIA and uncover a molecular mechanism CMV uses to manipulate host＇s attractiveness to insect vectors by targeting host hormone signaling.
Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. ...However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-∆2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-∆2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-UKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-∆2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-∆2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21-and 22-nucleotide secondary siRNAs are discussed.
As obligate intracellular phytopathogens, plant viruses must take advantage of hosts plasmodesmata and phloem vasculature for their local and long-distance transports to establish systemic infection ...in plants. In contrast to well-studied virus local transports, molecular mechanisms and related host genes governing virus systemic trafficking are far from being understood. Here, we performed a forward genetic screening to identify Arabidopsis thaliana mutants with enhanced susceptibility to a 2b-deleted mutant of cucumber mosaic virus (CMV-2aT∆2b). We found that an uncharacterized Piezo protein (AtPiezo), an ortholog of animal Piezo proteins with mechanosensitive (MS) cation channel activities, was required for inhibiting systemic infection of CMV-2aT∆2b and turnip mosaic virus tagged a green fluorescent protein (GFP) (TuMV-GFP). AtPiezo is induced by virus infection, especially in the petioles of rosette leaves. Thus, we for the first time demonstrate the biological function of Piezo proteins in plants, which might represent a common antiviral strategy because many monocot and dicot plant species have a single Piezo ortholog.
In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and ...slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5'-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3' half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.
Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect ...vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.
The recombinant NCMV‐RFP virus was rescued in barley plants and insect vectors to reveal cellular superinfection exclusion of two cytorhabdoviruses, NCMV‐RFP and BYSMV‐GFP, in barley.
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