Unveiling the Role of Reactive Oxygen Species and MicroRNAs in Plant Virus Interactions: A Focus on Bamboo Mosaic Virus

Virus infections often manifest as mosaic or mottling patterns on leaves, symptoms frequently linked to elevated levels of reactive oxygen species (ROS). While the connection between viral infection and ROS production is well-established, the precise role of ROS in symptom development remains an area of active investigation. This article delves into the intricate interplay between ROS, microRNAs (miRNAs), and viral infection, using Bamboo mosaic virus (BaMV) as a model system. BaMV, known for inducing chlorotic mosaic symptoms in plants like Brachypodium distachyon and Nicotiana benthamiana, provides a valuable platform for dissecting these complex interactions.

The Link Between BaMV Infection and Hydrogen Peroxide Accumulation

Bamboo mosaic virus (BaMV) causes chlorotic mosaic symptoms in both Brachypodium distachyon and Nicotiana benthamiana. To investigate the involvement of ROS in symptom development, researchers compared wild-type BaMV with a mutant version, BaMV△CPN35. The BaMV△CPN35 mutant, characterized by an N-terminal deletion in its coat protein gene, exhibits asymptomatic infection regardless of virus titer. Histochemical staining revealed a striking difference: hydrogen peroxide (H2O2) accumulated specifically in the chlorotic spots induced by BaMV, but not in leaves infected with the asymptomatic BaMV△CPN35 mutant or in mock-infected leaves. Moreover, exogenous H2O2 treatment enhanced yellowish chlorosis in BaMV-infected leaves. This finding suggests a direct link between H2O2 accumulation and the development of chlorotic symptoms in BaMV-infected plants.

Modulation of Superoxide Dismutase Expression by BaMV

Plants possess a sophisticated arsenal of antioxidant enzymes to mitigate the damaging effects of ROS. Among these, superoxide dismutases (SODs) play a crucial role in converting superoxide anions (O.2−) into H2O2, a less reactive ROS. Both BaMV and BaMV△CPN35 infections were found to induce the expression of Cu/Zu superoxide dismutase (CSD) antioxidants at both the messenger RNA and protein level. However, a key difference emerged in the processing of NbCSD2, a specific CSD isoform in N. benthamiana. BaMV infection triggered the abundant accumulation of full-length NbCSD2 preprotein (prNbCSD2, without transit peptide cleavage), whereas BaMV△CPN35 induced a truncated prNbCSD2. This differential processing suggests that BaMV infection may interfere with the proper targeting of NbCSD2 to its intended location within the cell.

Subcellular Localization of NbCSD2: A Tale of Two Viruses

To further investigate the fate of NbCSD2 during BaMV infection, researchers employed confocal microscopy to track the localization of NbCSD2 fused to green fluorescent protein (GFP). In cells infected with BaMV, the majority of NbCSD2-GFP was found to reside in the cytosol, the main compartment of the cell, with limited presence in chloroplasts. In contrast, BaMV△CPN35 infection tended to cause NbCSD2-GFP to remain in chloroplasts, the organelles where photosynthesis takes place. This observation suggests that BaMV infection disrupts the normal trafficking of NbCSD2, potentially impairing its ability to scavenge superoxide within chloroplasts.

The Role of miR398 in Regulating CSD Expression and BaMV Accumulation

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a critical role in regulating gene expression in plants. These molecules act by binding to messenger RNAs (mRNAs), leading to their degradation or translational repression. To explore the potential involvement of miRNAs in BaMV infection, researchers focused on miR398, a well-known stress-responsive miRNA that targets CSDs. Using 5′-RNA ligase-mediated rapid amplification of cDNA ends, the team validated that CSDs are indeed targets of miR398 in vivo. Furthermore, they found that BaMV infection increased the level of miR398, suggesting that the virus may be manipulating miRNA expression to its advantage. Interestingly, the level of BaMV titer was regulated positively by miR398 but negatively by CSD2. In contrast, overexpression of cytosolic form NbCSD2, impairing the transport into chloroplasts, greatly enhanced BaMV accumulation.

Plants' Response to Stress: The Role of ROS and Chloroplasts

Plants have evolved sophisticated mechanisms to sense and respond to unfavorable environmental cues. Production of reactive oxygen species (ROS) is a common plant response to various biotic and abiotic stresses. As a very dynamic signaling compartment, chloroplasts can sense biotic and abiotic perturbations to produce pro-defense molecules, including hormones such as salicylic, jasmonic and abscisic acids (ABAs), as well as secondary messengers (calcium and ROS). Typically, virus infection rapidly increases ROS levels (termed an oxidative burst), including hydroxyl radicals (.OH), superoxide anions (O.2−) and hydrogen peroxide (H2O2). SUPEROXIDE DISMUTASES (SODs) are important ROS scavengers in plant cells that convert O.2− to H2O2 in the first step of the detoxifying process during pathogen-induced oxidative burst. The balance between increased oxidative stress and antioxidant levels mediates symptom development in systemically virus-infected plants.

MicroRNAs: Fine-Tuning Stress Responses in Plants

Studies have shown that stress-responsive genes are tightly regulated and fine-tuned by a group of small RNAs (sRNAs), termed microRNAs (miRNAs). Plant miRNAs are a class of endogenous sRNAs of 20-24 nt that modulate biological and developmental events by negatively regulating gene expression via degradation or translational repression of messenger RNAs (mRNAs). In Arabidopsis thaliana, primary miRNAs with characteristic imperfect stem-loop or hairpin structures are transcribed by RNA polymerase II from MIR genes and then catalyzed by DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), DOUBLE-STRANDED RNA-BINDING PROTEIN (DRB) 1/2, and SERRATE (SE) to generate precursor miRNAs (pre-miRNAs). Pre-miRNAs are processed into a 20- to 24-nt miRNA duplex by DCL1, HYL1, and SE, and then methylated at the 3′-terminus by HUA ENHANCER 1, before being exported into the cytoplasm by HASTY 1. In general, miRNA-5p (from the 5′-arm) of the miRNA duplex loads into AGRONAUTE 1 (AGO1) to form an active RNA-induced silencing complex (RISC), which guides RISC to bind target transcripts of complementary sequence. Virus infection alters the profile of sRNAs by generating virus-derived sRNAs and altering endogenous sRNAs, including miRNA accumulation. Virus infection can even activate miRNA gene transcription. Constitutive expression of VIRAL SUPPRESSOR OF RNA SILENCING was able to alter miRNA levels and activity. Apart from miRNAs, a class of virus-activated host-encoded small RNAs (vasiRNAs) may also be induced by RNA virus infection, with some vasiRNAs having been characterized to target genes important in plant immunity.

The Complex Role of miRNAs in Virus Infection

Studies in animal systems have shown that miRNAs may directly target RNA viruses to restrict or, rather surprisingly, enhance infection. As yet, there is no direct evidence showing direct binding of miRNAs to plant viral genomes, except for some in silico analyses. However, it has been demonstrated that plant RNA viruses can hijack miRNAs to suppress innate immunity, supporting the notion that viruses manipulate miRNAs to generate a more permissive environment for viral accumulation.

miR398: A Key Player in Stress Response and Virus Interactions

miR398 is one of the best studied stress-responsive miRNAs and it is evolutionarily well-conserved across the plant kingdom. miR398-3p accumulation is mediated by various abiotic and biotic stresses including salinity, ABA, light, heat, sucrose, copper and iron concentrations, nitrogen deficiency, oxidative stress, and infection by bacteria, fungi, or viruses. Arabidopsis thaliana possesses three miR398-type miRNAs: miR398a, miR398b, and miR398c. miR398-3p targets cytosolic (CSD1) and chloroplastic (CSD2) forms of the Cu/Zu SOD, COPPER CHAPERONE FOR SUPEROXIDE DISMUTASE (CCS), MITOCHONDRIAL CYTOCHROME OXIDASE SUBUNIT V (COX5), BLUE COPPER-BINDING PROTEIN (BCBP), and an incompletely annotated plastocyanin-like domain-containing protein, of which the expression is altered according to changing miR398 levels. miR398b,c regulate CSD2 and CCS via mRNA cleavage and translational repression, but CSD1, COX5, and BCBP expression is regulated by miR398 at the mRNA level. It is also known that miR398b,c is transcribed by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 transcription factor under copper-limiting conditions, but CSD2 transcription is unaffected by the presence or absence of copper. Although multiple studies have shown that miR398 hyper-accumulates in various virus-infected plant species, the biological roles of this group of miRNAs remain unclear. Recently, it was reported that lethal systemic necrosis in potato spindle tuber viroid-infected DCL2/4-knockdown transgenic Solanum lycopersicum is accompanied by miR398 upregulation and ROS overproduction.

Bamboo Mosaic Virus: A Model for Studying Virus-Host Interactions

Bamboo mosaic virus (BaMV), a potexvirus of the alphavirus-like superfamily, contains a single-stranded, positive-sense RNA genome with a 5′-7mGpppG cap structure and a 3′-poly(A) tail. The viral genome comprises 6,400 nt (excluding the 3′-poly(A) tail), with five conserved open reading frames (ORFs) flanked by 5′- and 3′-untranslated regions (UTRs) of 94 and 142 nt, respectively. ORF 1 encodes a 155-kDa replicase containing three functional domains, i.e. methyltransferase, helicase, and RdRp domains. ORFs 2-4 are triple gene block protein (TGBp) genes that encode TGBp1-3 of 28, 13, and 6 kDa, respectively. All three TGBps are required for BaMV cell-to-cell movement. ORF 5 encodes the 25-kDa coat protein (CP), which is expressed by a 1.0-kb subgenomic RNA. BaMV CP is involved in virus encapsidation, cell-to-cell movement, and also a symptom determinant in BaMV-infected N. benthamiana. The BaMV△CPN35 mutant, in which the N-terminal 35 amino acids (aa) of the CP gene have been deleted, results in asymptomatic infection in N. benthamiana, independently of viral titer. In addition, BaMV can induce symptomless infection in A.

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Unraveling the Role of miR398 in BaMV Accumulation and Symptom Formation

In this study, researchers used wild-type (WT) symptomatic BaMV and asymptomatic BaMV△CPN35 mutant to address the possible role of miR398 in virus accumulation and symptom formation. They found that miR398 is a positive regulator for BaMV accumulation but its target NbCSD2 is a negative regulator. Both WT BaMV and BaMV△CPN35 infection could induce NbCSD level. However, only WT BaMV infection triggered unprocessed full-length NbCSD2 resided in the cytoplasm and triggered high level accumulation of H2O2, thereby resulting in chlorotic symptoms in BaMV-infected N. BaMV infection causes yellowish mosaic and chlorotic symptoms in N. benthamiana, with the N-terminal 35 aa of BaMV CP being symptom determinant. In Brachypodium distachyon, BaMV induces brown stripes and some light green spots in inoculated and systemic leaves (SLs). In contrast, BaMV△CPN35 infection is symptomless in these two plants. Typically, ROS accumulation and enhanced antioxidant levels accompany symptom development in compatible virus-plant interactions.

Investigating ROS Accumulation in BaMV-Infected Plants

Researchers ILs of N. benthamiana and B. distachyon with BaMV and BaMV△CPN35 virions and then harvested them at 10-d post-inoculation (dpi) when BaMV infection had induced mosaic symptoms. In situ histochemical staining of superoxide and H2O2 revealed that superoxide distribution was not associated with BaMV-induced symptoms. Superoxide levels were higher in mock-treated relative to BaMV- or BaMV△CPN35- ILs. However, H2O2 only accumulated in symptomatic regions of BaMV- but not BaMV△CPN35-infected IL and SLs of both N. benthamiana and B. distachyon. Thus, H2O2 localized in the yellowish mosaic spots of N. benthamiana and in the dark brown stripes of B. distachyon. H2O2 accumulated in BaMV-induced chlorotic tissues.

The Interplay Between Superoxide and Hydrogen Peroxide Levels

There are two possibilities why H2O2 accumulates in the chlorotic mosaic spots of BaMV-infected leaves: higher SOD activity or repressed photosynthesis, with this latter resulting in lower superoxide formation during photosynthetic electron transport in BaMV-infected plants compared to that in mock-infected plants. However, superoxide levels in BaMV- and BaMV△CPN35-inoculated N. benthamiana leaves were comparable, despite H2O2 levels being undetectable in BaMV△CPN35-ILs by in situ histochemical staining. These results imply that the higher H2O2 level in BaMV- than that in mock- or BaMV△CPN35-infected plants may be due to greater SOD activity. SODs can be classified into three groups: CSDs, iron SOD (FeSOD), and manganese SOD (MnSOD), which are localized in cytosol/chloroplasts, peroxisomes/chloroplasts, and mitochondria/peroxisomes, respectively. Superoxides are removed from chloroplasts by FeSOD and CSD.

Cloning and Characterization of NbCSD1 and NbCSD2

To confirm that result, researchers conducted a BLAST analysis on the AtCSD1 and AtCSD2 sequences against the N. They designed specific primers to clone full-length cDNAs of NbCSD1 and NbCSD2 by reverse transcription PCR (RT-PCR) and conducted 5′-rapid amplification of cDNA ends (RACE) on WT N. benthamiana. They found that the NbCSD1 and AtCSD1 proteins share 70% identity, whereas NbCSD2 and AtCSD2 proteins share 80% identity. They did not observe any difference in viral CP and TGBp1 levels between BaMV- and BaMV△CPN35-infected leaves by western blot. However, RNA and protein levels of NbCSD1 and NbCSD2 were increased in BaMV△CPN35-infected leaves, and slightly more so in BaMV-infected leaves, relative to mock-infected leaves. Notably, NbCSD2 was undetectable in mock-infected plants. However, rather than mature NbCSD2 (i.e. lacking transit peptide, ∼20 kDa), they detected NbCSD2 preprotein (∼31 kDa, prNbCSD2) or a truncated prNbCSD2 (∼28 kDa) using anti-CSD2 in BaMV- or BaMV△CPN35-infected leaves, respectively. Thus, BaMV and BaMV△CPN35 infection induces expression of NbCSD1 and NbCSD2 in infected N.

Differential Expression of NbCSD1 and NbCSD2 Induced by BaMV and BaMV△CPN35

NbCSD1 and NbCSD2 levels were analyzed by RT-qPCR, and BaMV TGBp1 and CP, NbCSD1 and NbCSD2 expression were analyzed by protein blot in the ILs at 5 dpi in mock-, BaMV-, and BaMV△CPN35-infected N. benthamiana. BaMV infection induced accumulation of prNbCSD2 rather than mature NbCSD2. Researchers hypothesized that this outcome was due to failed targeting of NbCSD2 into chloroplasts. Therefore, they generated a CSD2- green fluorescent protein (GFP) construct and coinfiltrated it with BaMV or BaMV△CPN35 to visualize the subcellular localization of NbCSD2. They used CSD2noTP-GFP harboring mutations at two positively charged residues within the transit peptide as a control. As expected, in mock-infiltrated cells, CSD2-GFP localized solely in chloroplasts, whereas CSD2noTP-GFP resided in both cytosol and chloroplasts. However, in BaMV-infected cells, CSD2-GFP signal mostly emanated from cytosol at the cell periphery and was limited from chloroplasts. In contrast, in BaMV△CPN35-infected cells, CSD2-GFP signal was mostly associated with chloroplasts and much less so in …

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