Autophagy plays an antiviral defence role against tomato spotted wilt orthotospovirus and is counteracted by viral effector NSs.

Autophagy, an intracellular degradation process, has emerged as a crucial innate immune response against various plant pathogens, including viruses. Tomato spotted wilt orthotospovirus (TSWV) is a highly destructive plant pathogen that infects over 1000 plant species and poses a significant threat to global food security. However, the role of autophagy in defence against the TSWV pathogen, and whether the virus counteracts this defence, remains unknown. In this study, we report that autophagy plays an important role in antiviral defence against TSWV infection; however, this autophagy-mediated defence is counteracted by the viral effector NSs. Transcriptome profiling revealed the up-regulation of autophagy-related genes (ATGs) upon TSWV infection. Blocking autophagy induction by chemical treatment or knockout/down of ATG5/ATG7 significantly enhanced TSWV accumulation. Notably, the TSWV nucleocapsid (N) protein, a major component of the viral replication unit, strongly induced autophagy. However, the TSWV nonstructural protein NSs was able to effectively suppress N-induced autophagy in a dose-dependent manner. Further investigation revealed that NSs inhibited ATG6-mediated autophagy induction. These findings provide new insights into the defence role of autophagy against TSWV, a representative segmented negative-strand RNA virus, as well as the tospoviral pathogen counterdefence mechanism.

Genome-wide analysis of tobacco NtTOM1/TOM3 gene family and identification of NtTOM1a/ NtTOM3a response to tobacco mosaic virus.

BACKGROUND: TOBAMOVIRUS MULTIPLICATION 1 (TOM1) and its homolog TOBAMOVIRUS MULTIPLICATION 3 (TOM3) play a prominent role in the multiplication of tobacco mosaic virus (TMV) in higher plants. Although homologs of NtTOM1/TOM3 genes have been identified in several plant species, little is known about the characteristics and functions of NtTOM1/TOM3 at the genome-wide level in tobacco (Nicotiana tabacum L.). RESULTS: In this study, we performed genome-wide identification and expression pattern analysis of the tobacco NtTOM1/TOM3 gene family. Twelve NtTOM1/TOM3 genes were identified and classified into four groups based on phylogenetic analysis. Sequence and conserved domain analyses showed that all these genes contained a specific DUF1084 domain. Expression pattern analysis showed that NtTOM1a, NtTOM1b, NtTOM1d, NtTOM3a, NtTOM3b, and NtTOM3d were induced by TMV at 1-, 3-, and 9 dpi, whereas the expression of other genes was not responsive to TMV at the early infection stage. TMV virion accumulation showed no obvious difference in either nttom1a or nttom3a mutants compared with the wild type. However, the virus propagation was significantly, but not completely, inhibited in the nttom1atom3a double mutant, indicating that other gene family members may function redundantly, such as NtTOM1b and NtTOM1d. In addition, overexpression of NtTOM1a or NtTOM3a also inhibited the TMV replication to some extent. CONCLUSIONS: The present study performed genome-wide analysis of the NtTOM1/TOM3 gene family in tobacco, and identified NtTOM1a and NtTOM3a as important genes involved in TMV multiplication based on functional analysis. These results provide a theoretical basis for further improving TMV resistance in tobacco.

Generation of dengue 3 envelope domain III using tobacco mosaic virus-based vector system and its immunological response mouse model by generating anti-dengue virus antibodies.

Producing recombinant proteins in plants has become a valuable alternative to traditional microbial or mammalian systems due to its cost-effectiveness, scalability, and ability to perform post-translational modifications. This study investigates the use of the Tobacco Mosaic Virus (TMV)-based vector system for producing the Dengue virus serotype 3 (DENV-3) envelope domain III (EDIII) protein in plants.. A fragment of the gene that encodes domain III of the dengue 3 envelope protein (D3EIII, comprising 300-420 amino acids), was effectively expressed within Nicotiana tabacum plants utilizing a transient expression system based on tobacco mosaic virus (TMV). The N-terminal 5' UTR region upstream of D3EIII notably enhanced protein yield in infected tissues. The produced recombinant protein exhibited reactivity with both (anti) D3EIII polyclonal antibodies and antibodies of anti-His tag. Upon injection of EDIII in mice, it stimulated the generation of antibodies against the dengue-specific virus. The induced antibodies demonstrated neutralizing activity against dengue virus type 3. These findings indicate that the TMV expression system is effective for producing dengue virus antigens in plants, resulting in antigens with appropriate properties and strong immunogenic potential.

Nanomaterial inactivates environmental virus and enhances plant immunity for controlling tobacco mosaic virus disease.

Tobacco mosaic virus (TMV) is extremely pathogenic and resistant to stress There are great needs to develop methods to reduce the virus in the environment and induce plant immunity simultaneously. Here, we report a multifunctional nano-protectant to reduce the virus in the environment and induce plant immunity simultaneously. The star polycation (SPc) nanocarrier can act as an active ingredient to interact with virus coat protein via electrostatic interaction, which reduces the proportion of TMV particles to 2.9% and leads to a reduction of the amount of virus in the environment by half. SPc can act as an adjuvant to spontaneously assemble with an immune inducer lentinan (LNT) through hydrogen bonding into nanoscale (142 nm diameter) LNT/SPc complex, which improves the physicochemical property of LNT for better wetting performance on leaves and cellular uptake, and further activates plant immune responses. Finally, the LNT/SPc complex displays preventive and curative effects on TMV disease, reducing TMV-GFP relative expression by 26% in the laboratory and achieving 82% control efficacy in the field We hope the strategy reported here would be useful for control of crop virus disease.

Synthesis, Anti-TMV Activities, and Action Mechanisms of a Novel Cytidine Peptide Compound.

Cytidine has a broad range of applications in the pharmaceutical field as an intermediate of antitumor or antiviral agent. Here, a series of new cytidine peptide compounds were synthesized using cytidine and Boc group-protected amino acids and analyzed for their antiviral activities against tobacco mosaic virus (TMV). Among these compounds, the structure of an effective antiviral cytidine peptide SN11 was characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometer. The compound SN11 has a molecular formula of C15H22N6O8 and is named 2-amino-N-(2- ((1- (3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-yl) amino) -2-oxyethyl) amino). The protection, inactivation, and curation activities of SN11 at a concentration of 500 µg/mL against TMV in Nicotiana glutinosa were 82.6%, 84.2%, and 72.8%, respectively. SN11 also effectively suppressed the systemic transportation of a recombinant TMV carrying GFP reporter gene (p35S-30B:GFP) in Nicotiana benthamiana by reducing viral accumulation to 71.3% in the upper uninoculated leaves and inhibited the systemic infection of TMV in Nicotiana tabacum plants. Furthermore, the results of RNA-seq showed that compound SN11 induced differential expression of genes involved in the biogenesis and function of ribosome, plant hormone signal transduction, plant pathogen interaction, and chromatin. These results validate the antiviral mechanisms of the cytidine peptide compound and provide a theoretical basis for their potential application in the management of plant virus diseases.

Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

Resistance of the CRISPR-Cas13a Gene-Editing System to Potato Spindle Tuber Viroid Infection in Tomato and Nicotiana benthamiana.

Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures.

Conservation of molecular responses upon viral infection in the non-vascular plant Marchantia polymorpha.

After plants transitioned from water to land around 450 million years ago, they faced novel pathogenic microbes. Their colonization of diverse habitats was driven by anatomical innovations like roots, stomata, and vascular tissue, which became central to plant-microbe interactions. However, the impact of these innovations on plant immunity and pathogen infection strategies remains poorly understood. Here, we explore plant-virus interactions in the bryophyte Marchantia polymorpha to gain insights into the evolution of these relationships. Virome analysis reveals that Marchantia is predominantly associated with RNA viruses. Comparative studies with tobacco mosaic virus (TMV) show that Marchantia shares core defense responses with vascular plants but also exhibits unique features, such as a sustained wound response preventing viral spread. Additionally, general defense responses in Marchantia are equivalent to those restricted to vascular tissues in Nicotiana, suggesting that evolutionary acquisition of developmental innovations results in re-routing of defense responses in vascular plants.

Comparative analysis of RNA interference and pattern-triggered immunity induced by dsRNA reveals different efficiencies in the antiviral response to potato virus X.

Antiviral responses induced by double-stranded RNA (dsRNA) include RNA interference (RNAi) and pattern-triggered immunity (PTI), but their relative contributions to antiviral defence are not well understood. We aimed at testing the impact of exogenous applied dsRNA on both layers of defence against potato virus X expressing GFP (PVX-GFP) in Nicotiana benthamiana. Co-inoculation of PVX-GFP with either sequence-specific (RNAi) or nonspecific dsRNA (PTI) showed that nonspecific dsRNA reduced virus accumulation in both inoculated and systemic leaves. However, nonspecific dsRNA was a poor inducer of antiviral immunity compared to a sequence-specific dsRNA capable of triggering the RNAi response, and plants became susceptible to systemic infection. Studies with a PVX mutant unable to move from cell to cell indicated that the interference with PVX-GFP triggered by nonspecific dsRNA operated at the single-cell level. Next, we performed RNA-seq analysis to examine similarities and differences in the transcriptome triggered by dsRNA alone or in combination with viruses harbouring sequences targeted or not by dsRNA. Enrichment analysis showed an over-representation of plant-pathogen signalling pathways, such as calcium, ethylene and MAPK signalling, which are typical of antimicrobial PTI. Moreover, the transcriptomic response to the virus targeted by dsRNA had a greater impact on defence than the non-targeted virus, highlighting qualitative differences between sequence-specific RNAi and nonspecific PTI responses. Together, these results further our understanding of plant antiviral defence, particularly the contribution of nonspecific dsRNA-mediated PTI. We envisage that both sequence-specific RNAi and nonspecific PTI pathways may be triggered via topical application of dsRNA, contributing cumulatively to plant protection against viruses.

Biosynthesized silver nanoparticles mediated by Ammi visnaga extract enhanced systemic resistance and triggered multiple defense-related genes, including SbWRKY transcription factors, against tobacco mosaic virus infection.

BACKGROUND: Tobacco mosaic virus (TMV) is a highly infectious plant virus that affects a wide variety of plants and reduces crop yields around the world. Here, we assessed the effectiveness of using Ammi visnaga aqueous seed extract to synthesize silver nanoparticles (Ag-NPs) and their potential to combat TMV. Different techniques were used to characterize Ag-NPs, such as scanning and transmission electron microscopy (SEM, TEM), energy-dispersive X-ray spectroscopy (EDS), fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). RESULTS: TEM demonstrated that the synthesized Ag-NPs had a spherical form with an average size of 23-30 nm and a zeta potential value of -15.9 mV, while FTIR revealed various functional groups involved in Ag-NP stability and capping. Interestingly, the Pre-treatment of tobacco plants (protective treatment) with Ag-NPs at 100-500 µg/mL significantly suppressed viral symptoms, while the Post-treatment (curative treatment) delayed their appearance. Furthermore, protective and curative treatments significantly increased chlorophyll a and b, total flavonoids, total soluble carbohydrates, and antioxidant enzymes activity (PPO, POX and CAT). Simultaneously, the application of Ag-NPs resulted in a decrease in levels of oxidative stress markers (H2O2 and MDA). The RT-qPCR results and volcano plot analysis showed that the Ag-NPs treatments trigger and regulate the transcription of ten defense-related genes (SbWRKY-1, SbWRKY-2, JERF-3, GST-1, POD, PR-1, PR-2, PR-12, PAL-1, and HQT-1). The heatmap revealed that GST-1, the primary gene involved in anthocyanidin production, was consistently the most expressed gene across all treatments throughout the study. Analysis of the gene co-expression network revealed that SbWRKY-19 was the most central gene among the studied genes, followed by PR-12 and PR-2. CONCLUSIONS: Overall, the reported antiviral properties (protective and/or curative) of biosynthesized Ag-NPs against TMV lead us to recommend using Ag-NPs as a simple, stable, and eco-friendly agent in developing pest management programs against plant viral infections.

Different viral effectors hijack TCP17, a key transcription factor for host Auxin synthesis, to promote viral infection.

Auxin is an important class of plant hormones that play an important role in plant growth development, biotic stress response, and viruses often suppress host plant auxin levels to promote infection. However, previous research on auxin-mediated disease resistance has focused mainly on signaling pathway, and the molecular mechanisms of how pathogenic proteins manipulate the biosynthetic pathway of auxin remain poorly understood. TCP is a class of plant-specific transcription factors, of which TCP17 is a member that binds to the promoter of YUCCAs, a key rate-limiting enzyme for auxin synthesis, and promotes the expression of YUCCAs, which is involved in auxin synthesis in plants. In this study, we reported that Tomato spotted wilt virus (TSWV) infection suppressed the expression of YUCCAs through its interaction with TCP17. Further studies revealed that the NSs protein encoded by TSWV disrupts the dimerization of TCP17, thereby inhibit its transcriptional activation ability and reducing the auxin content in plants. Consequently, this interference inhibits the auxin response signal and promotes the TSWV infection. Transgenic plants overexpressing TCP17 exhibit resistance against TSWV infection, whereas plants knocking out TCP17 were more susceptible to TSWV infection. Additionally, proteins encoded by other RNA viruses (BSMV, RSV and TBSV) can also interact with TCP17 and interfere with its dimerization. Notably, overexpression of TCP17 enhanced resistance against BSMV. This suggests that TCP17 plays a crucial role in plant defense against different types of plant viruses that use viral proteins to target this key component of auxin synthesis and promote infection.

Transient Expression Vector Construction, Subcellular Localisation, and Evaluation of Antiviral Potential of Flagellin BP8-2.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Structural Simplification of Podophyllotoxin: Discovery of γ-Butyrolactone Derivatives as Novel Antiviral Agents for Plant Protection.

Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 µg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin. Notably, the syn-diastereomer (syn-3g) exhibited superior antiviral activity compared to the anti-diastereomer (anti-3g). Mechanistic studies revealed that syn-3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g, with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.

A Negative Feedback Loop Compromises NMD-Mediated Virus Restriction by the Autophagy Pathway in Plants.

Nonsense-mediated decay (NMD) and autophagy play pivotal roles in restricting virus infection in plants. However, the interconnection between these two pathways in viral infections has not been explored. Here, it is shown that overexpression of NbSMG7 and NbUPF3 attenuates cucumber green mottle mosaic virus (CGMMV) infection by recognizing the viral internal termination codon and vice versa. NbSMG7 is subjected to autophagic degradation, which is executed by its interaction with one of the autophagy-related proteins, NbATG8i. Mutation of the ATG8 interacting motif (AIM) in NbSMG7 (SMG7mAIM1) abolishes the interaction and comprises its autophagic degradation. Silencing of NbSMG7 and NbATG8i, or NbUPF3 and NbATG8i, compared to silencing each gene individually, leads to more virus accumulations, but overexpression of NbSMG7 and NbATG8i fails to achieve more potent virus inhibition. When CGMMV is co-inoculated with NbSMG7mAIM1 or with NbUPF3, compared to co-inoculating with NbSMG7 in NbATG8i transgene plants, the inoculated plants exhibit milder viral phenotypes. These findings reveal that NMD-mediated virus inhibition is impaired by the autophagic degradation of SMG7 in a negative feedback loop, and a novel regulatory interplay between NMD and autophagy is uncovered, providing insights that are valuable in optimizing strategies to harness NMD and autophagy for combating viral infections.

Strigolactones Negatively Regulate Tobacco Mosaic Virus Resistance in Nicotiana benthamiana.

Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens but there are currently no reports on their role in the interaction between Nicotiana benthamiana and the tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, a SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. The KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.

Proviral role of ATG2 autophagy related protein in tomato bushy stunt virus replication through bulk phospholipid transfer into the viral replication organelle.

Subversion of cellular membranes and membrane proliferation are used by positive-strand RNA viruses to build viral replication organelles (VROs) that support virus replication. The biogenesis of the membranous VROs requires major changes in lipid metabolism and lipid transfer in infected cells. In this work, we show that tomato bushy stunt virus (TBSV) hijacks Atg2 autophagy related protein with bulk lipid transfer activity into VROs via interaction with TBSV p33 replication protein. Deletion of Atg2 in yeast and knockdown of Atg2 in Nicotiana benthamiana resulted in decreased TBSV replication. We found that subversion of Atg2 by TBSV was important to enrich VRO membranes with phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI(3)P phosphoinositide. Interestingly, inhibition of autophagy did not affect the efficient recruitment of Atg2 into VROs, and overexpression of Atg2 enhanced TBSV replication, indicating autophagy-independent subversion of Atg2 by TBSV. These findings suggest that the proviral function of Atg2 lipid transfer protein is in VRO membrane proliferation. In addition, we find that Atg2 interacting partner Atg9 with membrane lipid-scramblase activity is also coopted for tombusvirus replication. Altogether, the subversion of Atg2 bridge-type lipid transfer protein provides a new mechanism for tombusviruses to greatly expand VRO membranes to support robust viral replication.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.

Elucidating the Subcellular Localization of GLRaV-3 Proteins Encoded by the Unique Gene Block in N. benthamiana Suggests Implications on Plant Host Suppression.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3' proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry.

The Generation of Attenuated Mutants of East Asian Passiflora Virus via Deletion and Mutation in the N-Terminal Region of the HC-Pro Gene for Control through Cross-Protection.

East Asian Passiflora virus (EAPV) causes passionfruit woodiness disease, a major threat limiting passionfruit production in eastern Asia, including Taiwan and Vietnam. In this study, an infectious cDNA clone of a Taiwanese severe isolate EAPV-TW was tagged with a green fluorescent protein (GFP) reporter to monitor the virus in plants. Nicotiana benthamiana and yellow passionfruit plants inoculated with the construct showed typical symptoms of EAPV-TW. Based on our previous studies on pathogenicity determinants of potyviral HC-Pros, a deletion of six amino acids (d6) alone and its association with a point mutation (F8I, simplified as I8) were conducted in the N-terminal region of the HC-Pro gene of EAPV-TW to generate mutants of EAPV-d6 and EAPV-d6I8, respectively. The mutant EAPV-d6I8 caused infection without conspicuous symptoms in N. benthamiana and yellow passionfruit plants, while EAPV-d6 still induced slight leaf mottling. EAPV-d6I8 was stable after six passages under greenhouse conditions and displayed a zigzag pattern of virus accumulation, typical of a beneficial protective virus. The cross-protection effectiveness of EAPV-d6I8 was evaluated in both N. benthamiana and yellow passionfruit plants under greenhouse conditions. EAPV-d6I8 conferred complete cross-protection (100%) against the wild-type EAPV-TW-GFP in both N. benthamiana and yellow passionfruit plants, as verified by no severe symptoms, no fluorescent signals, and PCR-negative status for GFP. Furthermore, EAPV-d6I8 also provided complete protection against Vietnam's severe strain EAPV-GL1 in yellow passionfruit plants. Our results indicate that the attenuated mutant EAPV-d6I8 has great potential to control EAPV in Taiwan and Vietnam via cross-protection.