The effect of ectomycorrhizal fungal exposure on nursery-raised Pinus sylvestris seedlings: plant transpiration under short-term drought, root morphology and plant biomass.

Drought is a major environmental stressor that limits seedling growth. Several studies have found that some ectomycorrhizal fungi may increase the drought tolerance of nursery-raised seedlings. However, the precise role that different ectomycorrhizal fungi species play in drought tolerance remains unclear. We evaluated the transpiration rate of Pinus sylvestris seedlings under drought stress in greenhouse conditions by exposing seedlings to 10 ectomycorrhizal fungi species, with different functional traits (exploration type and hydrophobicity), and to 3 natural soil inoculums. We measured the transpiration and water potential of the seedlings during a 10-day drought period and a 14-day recovery period. We then analyzed their root morphology, stem, needle, root biomass and needle chlorophyll fluorescence. We showed that exposing seedlings to ectomycorrhizal fungi or soil inoculum had a positive effect on their transpiration rate during the driest period and through the recovery phase, leading to 2- to 3-fold higher transpiration rates compared with the nonexposed control seedlings. Seedlings exposed to medium-distance ectomycorrhizal fungi performed better than other exploration types under drought conditions, but ectomycorrhizal fungi hydrophobicity did not seem to affect the seedlings response to drought. No significant differences were observed in biomass accumulation and root morphology between the seedlings exposed to different ectomycorrhizal fungi species and the control. Our results highlight the positive and species-specific effect of ectomycorrhizal fungi exposure on drought tolerance in nursery-raised Scots pine seedlings. The studied ectomycorrhizal fungi functional traits may not be sufficient to predict the seedling response to drought stress, thus physiological studies across multiple species are needed to draw the correct conclusion. Our findings have potential practical implications for enhancing seedling drought tolerance in nursery plant production.

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana.

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus 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 losses 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 an 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 kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including 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.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. 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.

Development of FRET-based high-throughput screening for viral RNase III inhibitors.

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.

Phenotyping viral infection in sweetpotato using a high-throughput chlorophyll fluorescence and thermal imaging platform.

BACKGROUND: Virus diseases caused by co-infection with Sweet potato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) are a severe problem in the production of sweetpotato (Ipomoea batatas L.). Traditional molecular virus detection methods include nucleic acid-based and serological tests. In this study, we aimed to validate the use of a non-destructive imaging-based plant phenotype platform to study plant-virus synergism in sweetpotato by comparing four virus treatments with two healthy controls. RESULTS: By monitoring physiological and morphological effects of viral infection in sweetpotato over 29 days, we quantified photosynthetic performance from chlorophyll fluorescence (ChlF) imaging and leaf thermography from thermal infrared (TIR) imaging among sweetpotatoes. Moreover, the differences among different treatments observed from ChlF and TIR imaging were related to virus accumulation and distribution in sweetpotato. These findings were further validated at the molecular level by related gene expression in both photosynthesis and carbon fixation pathways. CONCLUSION: Our study validated for the first time the use of ChlF- and TIR-based imaging systems to distinguish the severity of virus diseases related to SPFMV and SPCSV in sweetpotato. In addition, we demonstrated that the operating efficiency of PSII and photochemical quenching were the most sensitive parameters for the quantification of virus effects compared with maximum quantum efficiency, non-photochemical quenching, and leaf temperature.

Potyviral Gene-Silencing Suppressor HCPro Interacts with Salicylic Acid (SA)-Binding Protein 3 to Weaken SA-Mediated Defense Responses.

The viral infection process is a battle between host defense response and pathogen antagonizing action. Several studies have established a tight link between the viral RNA silencing suppressor (RSS) and the repression of salicylic acid (SA)-mediated defense responses, nonetheless host factors directly linking an RSS and the SA pathway remains unidentified. From yeast two-hybrid analysis, we identified an interaction between the potyviral RSS helper-component proteinase (HCPro) and SA-binding protein SABP3. Co-localization and bimolecular fluorescence complementation analyses validated the direct in vivo interaction between Turnip mosaic virus (TuMV) HCPro and the Arabidopsis homologue of SABP3, AtCA1. Additionally, transient expression of TuMV HCPro demonstrated its ability to act as a negative regulator of AtCA1. When the plants of the AtCA1 knockout mutant line were inoculated with TuMV, our results indicated that AtCA1 is essential to restrict viral spreading and accumulation, induce SA accumulation, and trigger the SA pathway. Unexpectedly, the AtCA1 overexpression line also displayed a similar phenotype, suggesting that the constitutive expression of AtCA1 antagonizes the SA pathway. Taken together, our results depict AtCA1 as an essential regulator of SA defense responses. Moreover, the interaction of potyviral HCPro with this regulator compromises the SA pathway to weaken host defense responses and facilitate viral infection.

Family-based linkage and association mapping reveals novel genes affecting Plum pox virus infection in Arabidopsis thaliana.

Sharka is a devastating viral disease caused by the Plum pox virus (PPV) in stone fruit trees and few sources of resistance are known in its natural hosts. Since any knowledge gained from Arabidopsis on plant virus susceptibility factors is likely to be transferable to crop species, Arabidopsis's natural variation was searched for host factors essential for PPV infection. To locate regions of the genome associated with susceptibility to PPV, linkage analysis was performed on six biparental populations as well as on multiparental lines. To refine quantitative trait locus (QTL) mapping, a genome-wide association analysis was carried out using 147 Arabidopsis accessions. Evidence was found for linkage on chromosomes 1, 3 and 5 with restriction of PPV long-distance movement. The most relevant signals occurred within a region at the bottom of chromosome 3, which comprises seven RTM3-like TRAF domain-containing genes. Since the resistance mechanism analyzed here is recessive and the rtm3 knockout mutant is susceptible to PPV infection, it suggests that other gene(s) present in the small identified region encompassing RTM3 are necessary for PPV long-distance movement. In consequence, we report here the occurrence of host factor(s) that are indispensable for virus long-distance movement.