Reduction in vertical transmission rate of bean common mosaic virus in bee-pollinated common bean plants.

Vertical transmission, the transfer of pathogens across generations, is a critical mechanism for the persistence of plant viruses. The transmission mechanisms are diverse, involving direct invasion through the suspensor and virus entry into developing gametes before achieving symplastic isolation. Despite the progress in understanding vertical virus transmission, the environmental factors influencing this process remain largely unexplored. We investigated the complex interplay between vertical transmission of plant viruses and pollination dynamics, focusing on common bean (Phaseolus vulgaris). The intricate relationship between plants and pollinators, especially bees, is essential for global ecosystems and crop productivity. We explored the impact of virus infection on seed transmission rates, with a particular emphasis on bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV). Under controlled growth conditions, BCMNV exhibited the highest seed transmission rate, followed by BCMV and CMV. Notably, in the field, bee-pollinated BCMV-infected plants showed a reduced transmission rate compared to self-pollinated plants. This highlights the influence of pollinators on virus transmission dynamics. The findings demonstrate the virus-specific nature of seed transmission and underscore the importance of considering environmental factors, such as pollination, in understanding and managing plant virus spread.

A resilient mutualistic interaction between cucumber mosaic virus and its natural host to adapt to an excess zinc environment and drought stress.

A perennial pseudometallophyte Arabidopsis halleri is frequently infected with cucumber mosaic virus (CMV) in its natural habitat. The purpose of this study was to characterize the effect of CMV infection on the environmental adaptation of its natural host A. halleri. The CMV(Ho) strain isolated from A. halleri was inoculated into clonal virus-free A. halleri plants, and a unique plant-virus system consisting of CMV(Ho) and its natural wild plant host was established. In a control environment with ambient zinc supplementation, CMV(Ho) infection retarded growth in the above-ground part of host plants but conferred strong drought tolerance. On the other hand, in an excess zinc environment, simulating a natural edaphic environment of A halleri, host plants hyperaccumulated zinc and CMV(Ho) infection did not cause any symptoms to host plants while conferring mild drought tolerance. We also demonstrated in Nicotiana benthamiana as another host that similar effects were induced by the combination of excess zinc and CMV(Ho) infection. Transcriptomic analysis indicated that the host plant recognized CMV(Ho) as a mutualistic symbiont rather than a parasitic pathogen. These results suggest a resilient mutualistic interaction between CMV(Ho) and its natural host A. halleri in its natural habitat.

Plasmodesmal endoplasmic reticulum proteins regulate intercellular trafficking of cucumber mosaic virus in Arabidopsis.

Plasmodesmata (PD) are plasma membrane-lined cytoplasmic nanochannels that mediate cell-to-cell communication across the cell wall. A range of proteins are embedded in the PD plasma membrane and endoplasmic reticulum (ER), and function in regulating PD-mediated symplasmic trafficking. However, knowledge of the nature and function of the ER-embedded proteins in the intercellular movement of non-cell-autonomous proteins is limited. Here, we report the functional characterization of two ER luminal proteins, AtBiP1/2, and two ER integral membrane proteins, AtERdj2A/B, which are located within the PD. These PD proteins were identified as interacting proteins with cucumber mosaic virus (CMV) movement protein (MP) in co-immunoprecipitation studies using an Arabidopsis-derived plasmodesmal-enriched cell wall protein preparation (PECP). The AtBiP1/2 PD location was confirmed by TEM-based immunolocalization, and their AtBiP1/2 signal peptides (SPs) function in PD targeting. In vitro/in vivo pull-down assays revealed the association between AtBiP1/2 and CMV MP, mediated by AtERdj2A, through the formation of an AtBiP1/2-AtERdj2-CMV MP complex within PD. The role of this complex in CMV infection was established, as systemic infection was retarded in bip1/bip2w and erdj2b mutants. Our findings provide a model for a mechanism by which the CMV MP mediates cell-to-cell trafficking of its viral ribonucleoprotein complex.

Environmental Conditions Modulate Plant Virus Vertical Transmission and Survival of Infected Seeds.

Seed transmission is a major mode for plant virus persistence and dispersal, as it allows for virus survival within the seed in unfavorable conditions and facilitates spread when they become more favorable. To access these benefits, viruses require infected seeds to remain viable and germinate in altered environmental conditions, which may also be advantageous for the plant. However, how environmental conditions and virus infection affect seed viability, and whether these effects modulate seed transmission rate and plant fitness, is unknown. To address these questions, we utilized turnip mosaic virus, cucumber mosaic virus, and Arabidopsis thaliana as model systems. Using seeds from plants infected by these viruses, we analyzed seed germination rates, as a proxy of seed viability, and virus seed transmission rate under standard and altered temperature, CO2, and light intensity. With these data, we developed and parameterized a mathematical epidemiological model to explore the consequences of the observed alterations on virus prevalence and persistence. Altered conditions generally reduced overall seed viability and increased virus transmission rate compared with standard conditions, which indicated that under environmental stress, infected seeds are more viable. Hence, virus presence may be beneficial for the host. Subsequent simulations predicted that enhanced viability of infected seeds and higher virus transmission rate may increase virus prevalence and persistence in the host population under altered conditions. This work provides novel information on the influence of the environment in plant virus epidemics. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Design, synthesis, antiviral activity, and mechanisms of novel ferulic acid derivatives containing amide moiety.

To explore the novel compounds with high antiviral activity, three series ferulic acid derivatives containing amide moiety were gradually designed and synthesized based on antiviral activity tracking. The bioassay results exhibited that some target compounds had notable antiviral activities against tomato spotted wilt virus (TSWV) and cucumber mosaic virus (CMV). Compounds Y1, Y2, Y8, Z1 and Z2 presented splendid curative, protective, and inactivating activities to TSWV and CMV at 500 µg/mL. Especially, these compounds displayed outstanding inactivating effects on TSWV with the EC50 values of 225.9, 126.1, 224.6, 216.1, and 147.3 µg/mL, which were superior to ningnanmycin (249.1 µg/mL) and ribavirin (315.7 µg/mL). Furthermore, the antiviral mechanisms of compound Y2 were investigated by conducting microscale thermophoresis experiment and molecular docking experiment. The results suggested that compound Y2 performed excellent binding affinity to TSWV coat protein (TSWV CP) with the binding constant of 2.14 µM, which due to two strong hydrogen bonds of compound Y2 to the key amino acids ARG94 of TSWV CP. Therefore, compound Y2 can be regarded as a leading structure for development of the potential antiviral agent.

Incidence of RNA viruses infecting taro and tannia in East Africa and molecular characterisation of dasheen mosaic virus isolates.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) plants growing in 25 districts across Ethiopia, Kenya, Tanzania and Uganda were surveyed for four RNA viruses. Leaf samples from 392 plants were tested for cucumber mosaic virus (CMV), dasheen mosaic virus (DsMV), taro vein chlorosis virus (TaVCV) and Colocasia bobone disease-associated virus (CBDaV) by RT-PCR. No samples tested positive for TaVCV or CBDaV, while CMV was only detected in three tannia samples with mosaic symptoms from Uganda. DsMV was detected in 40 samples, including 36 out of 171 from Ethiopia, one out of 94 from Uganda and three out of 41 from Tanzania, while none of the 86 samples from Kenya tested positive for any of the four viruses. The complete genomes of nine DsMV isolates from East Africa were cloned and sequenced. Phylogenetic analyses based on the amino acid sequence of the DsMV CP-coding region revealed two distinct clades. Isolates from Ethiopia were distributed in both clades, while samples from Uganda and Tanzania belong to different clades. Seven possible recombination events were identified from the analysis carried out on the available 15 full-length DsMV isolates. Nucleotide substitution ratio analysis revealed that all the DsMV genes are under strong negative selection pressure.

Aphid endosymbiont facilitates virus transmission by modulating the volatile profile of host plants.

BACKGROUND: Most plant viruses rely on vectors for their transmission and spread. One of the outstanding biological questions concerning the vector-pathogen-symbiont multi-trophic interactions is the potential involvement of vector symbionts in the virus transmission process. Here, we used a multi-factorial system containing a non-persistent plant virus, cucumber mosaic virus (CMV), its primary vector, green peach aphid, Myzus persicae, and the obligate endosymbiont, Buchnera aphidicola to explore this uncharted territory. RESULTS: Based on our preliminary research, we hypothesized that aphid endosymbiont B. aphidicola can facilitate CMV transmission by modulating plant volatile profiles. Gene expression analyses demonstrated that CMV infection reduced B. aphidicola abundance in M. persicae, in which lower abundance of B. aphidicola was associated with a preference shift in aphids from infected to healthy plants. Volatile profile analyses confirmed that feeding by aphids with lower B. aphidicola titers reduced the production of attractants, while increased the emission of deterrents. As a result, M. persicae changed their feeding preference from infected to healthy plants. CONCLUSIONS: We conclude that CMV infection reduces the B. aphidicola abundance in M. persicae. When viruliferous aphids feed on host plants, dynamic changes in obligate symbionts lead to a shift in plant volatiles from attraction to avoidance, thereby switching insect vector's feeding preference from infected to healthy plants.

Antiviral activity by lecithin-induced fengycin lipopeptides as a potent key substrate against Cucumber mosaic virus.

In this study, the effect of different growth substrates on the production of biosurfactants in the PPL strain of Bacillus amyloliquefaciens-a biocontrol agent for diseases affecting pepper and tomato plants-and on the antiviral effect of the PPL strain on Cucumber mosaic virus (CMV)-infected pepper plants was investigated. The multifunctional PPL strain exhibited enhanced growth and increased production of biosurfactants upon lecithin supplementation and consequently exhibited potent anti-CMV activity. The enhanced anti-CMV activity of the lecithin-supplemented PPL culture could be attributed to the antiviral effect as well as to the upregulation of plant defense-related genes. Treatment with pure commercial fengycins elicited a defense response against CMV in pepper plants; this effect was similar to that observed upon treatment with the lecithin-supplemented PPL culture. To the best of our knowledge, this is the first study to report the antiviral activity of lecithin-induced fengycin lipopeptides. These results suggest that the growth substrate affects antimicrobial production by B. amyloliquefaciens PPL, and consequently its antiviral activity.

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Structural changes induced by substitution of amino acid 129 in the coat protein of Cucumber mosaic virus.

Cucumber mosaic virus infection leads to mosaic symptoms on a broad range of crop plants. Mutation at positions 129 in the coat protein of virus causes alterations in the severity of symptoms caused by the viral infection. In our investigation, we performed long term molecular dynamics simulations to elucidate the effect of different amino acid substitutes (infectious and non-infectious) at position 129 in the coat protein of Cucumber mosaic virus using various structural parameters. We found that the contagious mutants displayed more flexibility at loops ßE-αEF (129-136) and ßF-ßG loop (155-163) as compared to the non-infectious and native structures. This specific study at the atomic level yields innovative ideas for designing new therapeutic agents against the pathogen, which would further pave the path for researchers to control this devastating plant virus.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Within-Host Multiplication and Speed of Colonization as Infection Traits Associated with Plant Virus Vertical Transmission.

Although vertical transmission from parents to offspring through seeds is an important fitness component of many plant viruses, very little is known about the factors affecting this process. Viruses reach the seed by direct invasion of the embryo and/or by infection of the ovules or the pollen. Thus, it can be expected that the efficiency of seed transmission would be determined by (i) virus within-host multiplication and movement, (ii) the ability of the virus to invade gametic tissues, (iii) plant seed production upon infection, and (iv) seed survival in the presence of the virus. However, these predictions have seldom been experimentally tested. To address this question, we challenged 18 Arabidopsis thaliana accessions with Turnip mosaic virus and Cucumber mosaic virus Using these plant-virus interactions, we analyzed the relationship between the effect of virus infection on rosette and inflorescence weights; short-, medium-, and long-term seed survival; virulence; the number of seeds produced per plant; virus within-host speed of movement; virus accumulation in the rosette and inflorescence; and efficiency of seed transmission measured as a percentage and as the total number of infected seeds. Our results indicate that the best estimators of percent seed transmission are the within-host speed of movement and multiplication in the inflorescence. Together with these two infection traits, virulence and the number of seeds produced per infected plant were also associated with the number of infected seeds. Our results provide support for theoretical predictions and contribute to an understanding of the determinants of a process central to plant-virus interactions.IMPORTANCE One of the major factors contributing to plant virus long-distance dispersal is the global trade of seeds. This is because more than 25% of plant viruses can infect seeds, which are the main mode of germplasm exchange/storage, and start new epidemics in areas where they were not previously present. Despite the relevance of this process for virus epidemiology and disease emergence, the infection traits associated with the efficiency of virus seed transmission are largely unknown. Using turnip mosaic and cucumber mosaic viruses and their natural host Arabidopsis thaliana as model systems, we have identified the within-host speed of virus colonization and multiplication in the reproductive structures as the main determinants of the efficiency of seed transmission. These results contribute to shedding light on the mechanisms by which plant viruses disperse and optimize their fitness and may help in the design of more-efficient strategies to prevent seed infection.

Cucumber mosaic virus infection modulated the phytochemical contents of Passiflora edulis.

Cucumber mosaic virus (CMV) caused huge agricultural impact on Passiflora edulis. However, the interactions between CMV and P. edulis are poorly unknown, which lead to lack of prevention and control measures. In this study, we identified the infection of CMV in P. edulis through modern small RNA sequencing (sRNA-seq) technology combined with traditional electron microscope and polymerase chain reaction (PCR) methods. We also confirmed CMV infection adversely affected or modulated the contents of phytochemicals and further injured the development of P. edulis; inversely, P. edulis modulated its resistance to CMV stress by increasing the levels of secondary metabolites and the activities of antioxidant enzymes components. This is of significant importance to understand the interaction between virus infection and plant host.

The 2b protein and C-terminal region of the 2a protein indispensably facilitate systemic movement of cucumber mosaic virus in radish with supplementary function by either the 3a or the coat protein.

BACKGROUND: In Raphanus sativus (Japanese radish), strain D8 of cucumber mosaic virus (CMV-D8) establishes a systemic infection and induces mild mosaic on upper, non-inoculated leaves, whereas strain Y of CMV (CMV-Y) causes only a local infection in the inoculated leaves. Here, we further analyzed the specific viral factor(s) of CMV-D8 that is (are) indispensable for systemic infection in Japanese radish. METHODS: To identify which genomic RNA(s) is (are) involved in systemic infection in radish, we carried out a pseudorecombination analysis between CMV-D8 and CMV-Y. With recombination analyses between CMV-D8 and CMV-Y using mutant/recombinant RNA2s, chimeric and point-mutated RNA3s, we identified viral factors that are indispensable for systemic infection. RESULTS: Viral RNA2 and RNA3 of CMV-D8 facilitated efficient virus spread into the upper, non-inoculated plant tissues of radish (cv. Tokinashi), but not those of CMV-Y. Recombinant RNA2s demonstrated that the 2b protein (2b) and the C-terminus of the 2a protein (2a) of CMV-D8 have a crucial role in systemic infection. In addition, we used chimeric and point-mutated RNA3s to that Pro17 and Pro129 in the coat protein (CP) of CMV-D8 are involved in efficient systemic infection and that Ser51 in the 3a protein (3a) of CMV-D8 has positive effects on systemic spread. The results suggested that these viral factors facilitate systemic infection of CMV-D8 in Japanese radish. CONCLUSION: The C-terminal region of 2a, the entire region of 2b, and supplementary function of either Ser51 in 3a or Pro17/Pro 129 in CP confer systemic infectivity on CMV-D8 in radish. These results further elucidate the complex interaction of viral proteins of CMV to complete systemic infection as a host-specific manner.

Construction of a high-density genetic linkage map and identification of gene controlling resistance to cucumber mosaic virus in Luffa cylindrica (L.) Roem. based on specific length amplified fragment sequencing.

Luffa cylindrica L. is a cash crop which has important health, medicinal and industrial value, but no high saturation genetic map has been constructed owing to a lack of efficient markers. Furthermore, no genes were reportedly responsible for CMV resistance in Luffa spp. Specific length amplified fragment sequencing (SLAF-seq) is a valuable tool for large-scale discovery of markers and genetic mapping. The present study reported the construction of a high-density genetic map and the mapping of CMV resistant genes by using an F2 population of 130 individuals and their two inbred line parents. A total of 271.01 Mb pair-end reads were generated. 100,077 high-quality SLAFs were detected, and 7404 of them were polymorphic. Finally, 3701 of the polymorphic markers were selected for genetic map construction, and 13 linkage groups were generated. The map spanned 1518.56 cM with an average distance of 0.41 cM between adjacent markers. Based on the newly constructed high-density map, one gene located on chromosome 1 (100.072-100.457 cM) was identified to regulate CMV resistance in L. cylindrica. A gag-polypeptide of LTR copia-type retrotransposon was predicted as the candidate gene responsible for CMV resistance in L. cylindrica. The high-density genetic map and the CMV resistant gene mapped and predicted in this study will be useful in future research.

Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis.

Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.

LreEF1A4, a Translation Elongation Factor from Lilium regale, Is Pivotal for Cucumber Mosaic Virus and Tobacco Rattle Virus Infections and Tolerance to Salt and Drought.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4; however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1-3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein.

The effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5' or 3' half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5' mutants were serially passaged in N. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5' mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCE Recently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

Tissue culture and next-generation sequencing: A combined approach for detecting yam (Dioscorea spp.) viruses.

In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus, confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm.