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Viroporins are small, hydrophobic viral proteins that modify cellular membranes to form tiny pores for influx of ions and small molecules. Previously, viroporins were identified exclusively in vertebrate viruses. Recent studies have shown that both plant-infecting positive-sense single-stranded (+ss) and negative-sense single-stranded (-ss) RNA viruses also encode functional viroporins. These seminal discoveries not only advance our understanding of the distribution and evolution of viroporins, but also open up a new field of plant virus research., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pattern-triggered immunity is the first line of defense against infection by pathogens such as bacteria and fungi in plants, and this mechanism remains poorly defined in plant viruses. Double-stranded RNA (dsRNA) is an intermediate in the replication of plant RNA viruses, and is considered to be a conserved structure of plant viruses similar to pathogen-associated molecular pattern. Whether dsRNA is the elicitor that activates plant immunity in response to virus infection remains obscure. In this method, we use the cDNA of turnip mosaic virus genome as the template to in vitro synthesis of viral dsRNA and examine whether viral dsRNA could activate plant immunity in Arabidopsis thaliana, including MAPK kinase cascade and reactive oxygen burst. In order to provide some references for researchers studying dsRNA in terms of research methodology and experimental methods, we use western blot to measure MAPK kinase cascade and luminol-based assay to measure ROS burst in Arabidopsis thaliana treated by viral dsRNA., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vacuolar (H + )-PPases (VPs), are key regulators of active proton (H + ) transport across membranes using the energy generated from PPi hydrolysis. The VPs also play vital roles in plant responses to various abiotic stresses. Their functions in plant responses to pathogen infections are unknown. Here, we show that TaVP, a VP of wheat (Triticum aestivum) is important for wheat resistance to Chinese wheat mosaic virus (CWMV) infection. Furthermore, overexpression of TaVP in plants induces the activity of PPi hydrolysis, leading to plants cell death. A virus-derived small interfering RNA (vsiRNA-20) generated from CWMV RNA1 can regulate the mRNA accumulation of TaVP in wheat. The accumulation of vsiRNA-20 can suppress cell death induced by TaVP in a dosage-dependent manner. Moreover, we show that the accumulation of vsiRNA-20 can affect PPi hydrolysis and the concentration of H + in CWMV-infected wheat cells to create a more favorable cellular environment for CWMV replication. We propose that vsiRNA-20 regulates TaVP expression to prevent cell death and to maintain a weak alkaline environment in cytoplasm to enhance CWMV infection in wheat. This finding may be used as a novel strategy to minimize virus pathogenicity and to develop new antiviral stratagems., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection., (© 2019 Her Majesty the Queen in Right of Canada The Plant Journal © 2019 John Wiley & Sons Ltd and Society for Experimental Biology.)

Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti‐defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)‐infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR‐mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV‐infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA‐dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant–virus interaction. [ABSTRACT FROM AUTHOR]

Viruses encounter numerous host factors that facilitate or suppress viral infection. Although some host factors manipulated by viruses were uncovered, we have limited knowledge of the pathways hijacked to promote viral replication and activate host defense responses. Turnip mosaic virus (TuMV) is one of the most prevalent viral pathogens in many regions of the world. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach to characterize cellular protein changes in the early stages of infection of Nicotiana benthamiana by wild type and replication-defective TuMV. A total of 225 differentially accumulated proteins (DAPs) were identified (182 increased and 43 decreased). Bioinformatics analysis showed that a few biological pathways were associated with TuMV infection. Four upregulated DAPs belonging to uridine diphosphate-glycosyltransferase (UGT) family members were validated by their mRNA expression profiles and their effects on TuMV infection. NbUGT91C1 or NbUGT74F1 knockdown impaired TuMV replication and increased reactive oxygen species production, whereas overexpression of either promoted TuMV replication. Overall, this comparative proteomics analysis delineates the cellular protein changes during early TuMV infection and provides new insights into the role of UGTs in the context of plant viral infection. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (RT-qPCR) is a widely used method for studying alterations in gene expression upon infections caused by diverse pathogens such as viruses. Positive-sense single-stranded (ss(+)) RNA viruses form a major part of all known plant viruses, and some of them are damaging pathogens of agriculturally important crops. Analysis of gene expression following infection by ss(+) RNA viruses is crucial for the identification of potential anti-viral factors. However, viral infections are known to globally affect gene expression and therefore selection and validation of reference genes for RT-qPCR is particularly important. In this study, the expression of commonly used reference genes for RT-qPCR was studied in Nicotiana benthamiana following single infection by 11 ss(+) RNA viruses, including five tobamoviruses, four potyviruses, one potexvirus and one polerovirus. Stability of gene expression was analyzed in parallel by four commonly used algorithms: geNorm, NormFinder, BestKeeper, and Delta CT, and RefFinder was finally used to summarize all the data. The most stably expressed reference genes differed significantly among the viruses, even when those viruses were from the same genus. Our study highlights the importance of the selection and validation of reference genes upon different viral infections. [ABSTRACT FROM AUTHOR]

Rice stripe virus (RSV) is one of the most important viral pathogens of rice in East Asia. The transcription of RSV is initiated by cap-snatching, a mechanism shared by influenza virus (IFV). This lends the possibility that antiviral compounds targeting the cap-snatching of IFV, many of which have been commercially available, may inhibit RSV transcription. A convenient and inexpensive system allowing researchers to test this idea, however, has been unavailable to date. Here, we show that purified RSV performs transcription in vitro and the transcription was readily detectable by nested reverse transcription-polymerase chain reaction (RT-PCR). With this system, we tested the effects of 2,4-dioxo-4-phenylbutanoic acid (DPBA) and pimodivir, two well-known IFV cap-snatching inhibitors, as well as ribavirin, a broad-spectrum antiviral compound whose targets remain elusive. In reaction mixtures containing 2.5 ng/µL of purified RSV, DPBA and pimodivir abolished RSV transcription at a concentration of 10 and 100 µM, respectively. In contrast, no inhibitory effect was detected from ribavirin, even at a concentration as high as 400 µM. These results suggest that at least some cap-snatching inhibitors of IFV are inhibitory to RSV transcription. These compounds, which can be identified with the experimental system described here, may serve as starting points in developing antivirals against RSV or related plant viruses. [ABSTRACT FROM AUTHOR]

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mosaic virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway. Author summary: Jasmonic acid pathway has emerged as one of the predominant battlefields between plants and viruses. Several studies have indicated that, in addition to interfering with JA signaling, plant viruses can also affect JA biosynthesis, but the direct molecular links between them remain elusive. Here, we identify a highly conserved chloroplast protein cpSRP54 as a key positive regulator in JA biosynthesis and a common target for viruses belong to different genera. Through associating with cpSRP54 and inducing its degradation using the protein they encoded, the viruses can inhibit the cpSRP54-facilitated delivery of AOCs to the thylakoid membrane and manipulation of JA-mediated defense. This capability of viruses might define a novel and effective strategy against the antiviral JA pathway. [ABSTRACT FROM AUTHOR]

The apoplast is the extracellular space for signalling, nutrient transport, and plant–microbe interactions, but little is known about how plant viruses use the foliar apoplast. Proteomic analysis of the apoplasts isolated from potato virus X (PVX)‐infected Nicotiana benthamiana plants showed that the coat protein (CP) is the dominant viral component. The presence of the CP in the apoplast was confirmed by western blot, viral nucleic acid was detected by reverse transcription‐PCR and northern blot, and viral particles were observed by transmission electron microscopy (TEM). The apoplast from infected leaves was infectious if rubbed onto healthy leaves but not when infiltrated into them. The exosomes were separated from the apoplast fluid by high‐speed centrifugation and TEM showed that PVX particles were not associated with the exosomes. These results suggest that PVX virions are released to the N. benthamiana apoplast in a one‐way manner and do not share the bidirectional transport of exosomes. [ABSTRACT FROM AUTHOR]