Effects of an alphasatellite on life cycle of the nanovirus Faba bean necrotic yellows virus

Nanoviruses are plant viruses with a multipartite single-stranded DNA (ssDNA) genome. Alphasatellites are commonly associated with nanovirus infections, but their putative impact on their helper viruses is unknown. In this study, we investigated the role of subterranean clover stunt alphasatellite 1 (hereafter named SCSA 1) on various important traits of faba bean necrotic yellows virus (FBNYV) in its host plant Vicia faba and aphid vector Acyrthosiphon pisum , including disease symptoms, viral accumulation and transmission. The results indicate that SCSA 1 does not affect the symptom severity nor the overall FBNYV accumulation in V. faba, but changes the relative amounts of its different genomic segments. Moreover, the association of SCSA 1 with FBNYV increases the rate of plant-to-plant transmission by a process seemingly unrelated to simple increase of the viral accumulation in the vector. These results represent the first study on the impact of an alphasatellite on the biology of its helper nanovirus. They suggest that SCSA 1 may benefit FBNYV, but the genericity of this conclusion is discussed and questioned. Importance Alphasatellites are circular single stranded DNA molecules frequently found in association with natural isolates of nanoviruses and some geminiviruse, the two ssDNA plant infecting virus families. While the implications of alphasatellite presence in geminivirus infections are relatively well documented, comparable studies on alphasatellites associated with nanoviruses are not available. Here we confirm that subterranean clover stunt alphasatellite 1 affects different traits of its helper nanovirus, faba bean necrotic yellows virus, both in the host plant and aphid vector. We show that the frequencies of the virus segments change in the presence of alphasatellite, in both plant and vector. We also confirm that while within-plant virus load and symptom are not affected by alphasatellite, the presence of alphasatellite decreases within-aphid virus load, but significantly increases virus transmission rate, so may confer a possible evolutionary advantage for the helper virus.

Determinants of Persistent Patterns of Pepino Mosaic Virus Mixed Infections

Pepino mosaic virus (PepMV) has become a pandemic virus in tomato crops, causing important economic losses worldwide. In Spain, isolates of the EU and CH2 strains co-circulate, with PepMV-EU predominantly found in mixed infections. Simultaneous in planta mixed infections result in an asymmetric antagonism against PepMV-CH2, but the outcome of over-infections has never been tested. PepMV-EU and PepMV-CH2 time-lagged inoculations were performed, and viral accumulation was measured 10 days after challenge inoculation. PepMV-EU had a protective effect over PepMV-CH2; in contrast, the accumulation of PepMV-EU increased in plants pre-inoculated with PepMV-CH2 as compared to single infections. We also studied the effect of the type of infection on viral transmission. Independently of the nature of the infection (single or mixed), we observed a strong positive correlation between virus accumulation in the source plant and transmission, excluding mixed infection effects different than modulating viral accumulation. Finally, in order to determine the genetic variability of PepMV strains in single and mixed infections, a 430 nucleotide region was RT-PCR amplified from samples from a serial passages experiment and deep-sequenced. No significant differences were found in the number of nucleotide substitutions between single and mixed infections for PepMV-EU; in contrast, significant differences were found for PepMV-CH2, which was more variable in single than in mixed infections. Comparing PepMV-EU with PepMV-CH2, a higher nucleotide diversity was found for PepMV-CH2. Collectively, our data strongly suggest that PepMV mixed infections can impact the virus epidemiology by modulating in planta virus strain accumulation and diversification.

A predicted stem-loop in coat protein-coding sequencing of tobacco vein banding mosaic virus is required for efficient replication

Potyviral Coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon c574g575c576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon c574g575c576 to a574g575a576 or a574g575g576, but not c574g575a576, c574g575t576, or c574g575g576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that c574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive c-g base pairs (C576-G531, c532-g575, c574-g533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon c532g533c534 to A532G533A534 and A532G533G534, but not c532g533a534, c532g533t534, or c532g533g534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that c532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial passage inoculation, the accumulation of spontaneous mutant viruses was restored and codons A532G533A534, A532G533G534, a574g575a576, or a574g575g576 of mutants was separately changed to C532G533A534, C532G533G534, C574g575a576, or C574g575g576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive c532-g575 and c574-g533 base pairs played critical roles in TVBMV replication via maintaining the stability of stem-loop structure formed by nucleotides 531 to 576 of CP-coding sequence.

Metabolic Profile Discriminates and Predicts Arabidopsis Susceptibility to Virus under Field Conditions

As obligatory parasites, plant viruses alter host cellular metabolism. There is a lack of information on the variability of virus-induced metabolic responses among genetically diverse plants in a natural context with daily changing conditions. To decipher the metabolic landscape of plant-virus interactions in a natural setting, twenty-six and ten accessions of Arabidopsis thaliana were inoculated with Turnip mosaic virus (TuMV), in two field experiments over 2 years. The accessions were measured for viral accumulation, above-ground biomass, targeted and untargeted metabolic profiles. The phenotypes of the accessions ranged from susceptibility to resistance. Susceptible and resistant accessions were shown to have different metabolic routes after inoculation. Susceptible genotypes accumulate primary and secondary metabolites upon infection, at the cost of hindered growth. Twenty-one metabolic signatures significantly accumulated in resistant accessions whereas they maintained their growth as mock-inoculated plants without biomass penalty. Metabolic content was demonstrated to discriminate and be highly predictive of the susceptibility of inoculated Arabidopsis. This study is the first to describe the metabolic landscape of plant-virus interactions in a natural setting and its predictive link to susceptibility. It provides new insights on plant-virus interactions. In this undomesticated species and in ecologically realistic conditions, growth and resistance are in a permanent conversation.

In vitro identification and in vivo confirmation of inhibitors for SPCSV RNA silencing suppressor, a viral RNase III

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield loss all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide-docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out a kinetic-based HTS using fluorescence resonance energy transfer technology that isolated 112 compounds. These compounds were validated with dose-response assays including the kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta. In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases. Significance statement We report here a high-throughput inhibitor identification that targets a severe sweetpotato virus disease caused by co-infection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield loss. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

Metabolic profile discriminates and predicts Arabidopsis susceptibility to virus under field conditions

SummaryAs obligatory parasites, plant viruses alter host cellular metabolism. There is a lack of information on the variability of virus-induced metabolic responses among genetically diverse plants in a natural context with daily changing conditions. To decipher the metabolic landscape of plant-virus interactions in a natural setting, one hundred and thirty-two and twenty-six accessions of Arabidopsis thaliana were inoculated with Turnip mosaic virus (TuMV), in two field experiments over 2 years. The accessions were phenotyped for viral accumulation, above-ground biomass, targeted and untargeted metabolic profiles. The accessions revealed quantitative response to the virus, from susceptibility to resistance. Susceptible accessions accumulate primary and secondary metabolites upon infection, at the cost of hindered growth. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) revealed that the primary metabolites sucrose, glucose and glutamate discriminate susceptible and resistant accessions. Twenty-one metabolic signatures were found to significantly accumulate in resistant accessions whereas they maintained their growth at the same level as mock-inoculated plants without biomass penalty.Metabolic content was demonstrated to discriminate and to be highly predictive of the susceptibility of inoculated Arabidopsis. The PLS coefficient estimated in the training data set reveals, after cross-validation, a correlation of 0.61 between predicted and true viral accumulation. This study is the first to describe the metabolic landscape of plant-virus interactions in a natural setting and its predictive link to susceptibility. It reveals that, in this undomesticated species and in ecologically realistic conditions, growth and resistance are in a permanent conversation and provides new insights on plant-virus interactions.

Apoptosis in a Whitefly Vector Activated by a Begomovirus Enhances Viral Transmission

Of the approximately 1,100 known plant viruses, about one-third are DNA viruses that are vectored by insects. Plant virus infections often induce cellular and molecular responses in their insect vectors, which can, in many cases, affect the spread of viruses. However, the mechanisms underlying vector responses that affect virus accumulation and transmission are poorly understood. Here, we examined the role of virus-induced apoptosis in the transmission of begomoviruses, a group of single-stranded plant DNA viruses that are transmitted by whiteflies and cause extensive damage to many crops worldwide. We demonstrated that virus infection can induce apoptosis in the insect vector conferring protection to the virions from degradation, leading to enhanced viral accumulation and transmission to host plants. Our findings provide valuable clues for designing new strategies to block the transmission of insect-vectored plant viruses, particularly plant DNA viruses.

Multiomics analysis of tolerant interaction of potato with potato virus Y

Potato virus Y (PVY) is the most economically important viral pathogen of potato worldwide. Different potato cultivars react to the pathogen differently, resulting in resistant, tolerant or disease outcome of the interaction. Here we focus on tolerant interaction between potato cv. Désirée and PVYNTN. To capture the response in its full complexity, we analyzed the dynamic changes on multiple molecular levels, including transcriptomics, sRNAomics, degradomics, proteomics and hormonomics. The analysis was complemented by the measurements of viral accumulation, photosynthetic activity and phenotypisation of the symptoms. Besides cv. Désirée we also studied its transgenic counterpart depleted for the accumulation of salicylic acid (NahG-Désirée). This multiomics analysis provides better insights into the mechanisms leading to tolerant response of potato to viral infection and can be used as a base in further studies of plant immunity regulation.

Olive mild mosaic virus coat protein and p6 are suppressors of RNA silencing and their silencing confers resistance against OMMV

RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress it. Here the genome of Olive mild mosaic virus (OMMV) was screened for silencing suppressors using a green fluorescent based transient suppression assay. The full OMMV cDNA and 5 different OMMV open reading frames (ORFs) were cloned into Gateway binary destination vector pK7WG2, transformed into Agrobacterium tumefaciens C58C1 and agroinfiltrated into Nicotiana benthamiana 16C plants. Among all ORFs tested, CP and p6 showed suppressor activity, with CP showing a significant higher activity when compared to p6, yet lower than that of the full OMMV. This suggests that OMMV silencing suppression results from a complementary action of both CP and p6.Such discovery led to the use of those viral suppressors in the development of OMMV resistant plants through pathogen-derived resistance (PDR) based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a highly significant reduction in viral accumulation and symptom attenuation was observed as compared to that seen when each construct was used alone, and to the respective controls, thus showing clear effectiveness against OMMV infection. Data here obtained indicate that the use of both OMMV viral suppressors as transgenes is a very efficient and promising approach to obtain plants resistant to OMMV.ImportanceOMMV silencing suppressors were determined. Among all ORFs tested, CP and p6 showed suppressor activity, with CP showing a significant higher activity when compared to p6, yet lower than that of the full OMMV, suggesting a complementary action of both CP and p6 in silencing suppression.This is the first time that a silencing suppressor was found in a necrovirus and that two independent proteins act as silencing suppressors in a member of the Tombusviridae family.When silencing of both suppressors was achieved, a highly significant reduction in viral accumulation and symptom attenuation was observed as compared to that seen when each was used alone, thus showing clear effectiveness against OMMV infection. A high percentage of resistant plants was obtained (60%), indicating that the use of both OMMV viral suppressors as transgenes is a very efficient and promising approach to obtain plants resistant to OMMV.

Co-infection with a wheat rhabdovirus causes a reduction inMal de Río Cuarto virustiter in its planthopper vector

Mal de Río Cuarto virus(MRCV,Fijivirus,Reoviridae) causes one of the most important diseases in maize (Zea maysL.) in Argentina and has been detected in mixed infections with a rhabdovirus closely related to Maize yellow striate virus. In nature both viruses are able to infect maize and several grasses including wheat, and are transmitted in a persistent propagative manner byDelphacodes kuscheliFennah (Hemiptera: Delphacidae). This work describes the interactions between MRCV and rhabdovirus within their natural vector and the consequences of such co-infection regarding virus transmission and symptom expression. First- and third-instarD. kuschelinymphs were fed on MRCV-infected wheat plants or MRCV-rhabdovirus-infected oat plants, and two latency periods were considered. Transmission efficiency and viral load of MRCV-transmitting and non-transmitting planthoppers were determined by real-time quantitative polymerase chain reaction analysis (RTqPCR). Vector transmission efficiency was related to treatments (life stages at acquisition and latency periods). Nevertheless, no correlation between transmission efficiency and type of inoculum used to infect insects with MRCV was found. Treatment by third-instar nymphs 17 days after Acquisition Access Period was the most efficient for MRCV transmission, regardless of the type of inoculum. Plants co-infected with MRCV and rhabdovirus showed the typical MRCV symptoms earlier than plants singly infected with MRCV. The transmitting planthoppers showed significantly higher MRCV titers than non-transmitting insects fed on single or mixed inocula, confirming that successful MRCV transmission is positively associated with viral accumulation in the insect. Furthermore, MRCV viral titers were higher in transmitting planthoppers that acquired this virus from a single inoculum than in those that acquired the virus from a mixed inoculum, indicating that the presence of the rhabdovirus somehow impaired MRCV replication and/or acquisition. This is the first study about interactions between MRCV and a rhabdovirus closely related to Maize yellow striate virus in this insect vector (D. kuscheli), and contributes to a better understanding of planthopper–virus interactions and their epidemiological implications.