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Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non-dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self-interaction and DNA binding to the promoters of unfolded protein response (UPR)-related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

The molecular interactions between Polymyxa betae , the protist vector of sugar beet viruses, beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, and Beta vulgaris have not been extensively studied. Here, the transmission of BNYVV to sugar beet by P. betae zoospores was optimized using genetically characterized organisms. Molecular interactions of aviruliferous and viruliferous protist infection on sugar beet were highlighted by transcriptomic analysis. P. betae alone induced limited gene expression changes in sugar beet, as a biotrophic asymptomatic parasite. Most differentially expressed plant genes were down-regulated and included resistance gene analogs and cell wall peroxidases. Several enzymes involved in stress regulation, such as the glutathione- S -transferases, were significantly induced. With BNYVV, the first stages of the P. betae life cycle on sugar beet were accelerated with a faster increase of relative protist DNA level and an earlier appearance of sporangia and sporosori in plants roots. A clear activation of plant defenses and the modulation of genes involved in plant cell wall metabolism were observed. The P. betae transcriptome in the presence of BNYVV revealed induction of genes possibly involved in the switch to the survival stage. The interactions were different depending on the presence or absence of the virus. P. betae alone alleviates plant defense response, playing hide-and-seek with sugar beet and allowing for their mutual development. Conversely, BNYVV manipulates plant defense and promotes the rapid invasion of plant roots by P. betae . This accelerated colonization is accompanied by the development of thick-walled resting spores, supporting the virus survival. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The A-type of beet necrotic yellow vein virus (BNYVV) is widely distributed in Europe and is one of the major virus types causing rhizomania disease in sugar beet. The closely related P-type is mainly limited to a small region in France (Pithiviers). Both virus types possess four RNAs (RNA1-4), but the P-type harbours an additional fifth RNA species (RNA5). The P-type is associated with stronger disease symptoms and resistance-breaking of Rz1 , one of the two resistance genes which are used to control BNYVV infection. These characteristics are presumably due to the presence of RNA5, but experimental evidence for this is lacking. We generated the first infectious cDNA clone of BNYVV P-type to study its pathogenicity in sugar beet in comparison to a previously developed A-type clone. Using this tool, we confirmed the pathogenicity of the P-type clone in the experimental host Nicotiana benthamiana and two Beta species, B. macrocarpa and B. vulgaris . Independent of RNA5 , both the A- and the P-type accumulated in lateral roots and reduced the taproot weight of a susceptible sugar beet genotype to a similar extent. In contrast, only the P-type clone was able to accumulate a virus titre in an Rz1 -resistant variety whereas the A-type clone failed to infect this variety. The efficiency of the P-type to overcome Rz1 resistance was strongly associated with the presence of RNA5. Only a double resistant variety, harbouring Rz1 and Rz2, prevented an infection with the P-type. Reassortment experiments between the P- and A-type clones demonstrated that both virus types can exchange whole RNA components without losing the ability to replicate and to move systemically in sugar beet. Although our study highlights the close evolutionary relationship between the two virus types, we were able to demonstrate distinct pathogenicity properties that are attributed to the presence of RNA5 in the P-type.

Background: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups., Methods: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value., Results: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data., Conclusions: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial., (© 2022. The Author(s).)

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is characterized by excessive lateral root (LR) formation. Auxin-mediated degradation of Aux/IAA transcriptional repressors stimulates gene regulatory networks leading to LR organogenesis and involves several Aux/IAA proteins acting at distinctive stages of LR development. Previously, we showed that BNYVV p25 virulence factor interacts with BvIAA28, a transcriptional repressor acting at early stages of LR initiation. The evidence suggested that p25 inhibits BvIAA28 nuclear localization, thus, de-repressing transcriptional network leading to LR initiation. However, it was not clear whether p25 interacts with other Aux/IAA proteins. Here, by adopting bioinformatics, in vitro and in vivo protein interaction approaches we show that p25 interacts also with BvIAA2 and BvIAA6. Moreover, we confirmed that the BNYVV infection is, indeed, accompanied by an elevated auxin level in the infected LRs. Nevertheless, expression levels of BvIAA2 and BvIAA6 remained unchanged upon BNYVV infection. Mutational analysis indicated that interaction of p25 with either BvIAA2 or BvIAA6 requires full-length proteins as even single amino acid residue substitutions abolished the interactions. Compared to p25-BvIAA28 interaction that leads to redistribution of BvIAA28 into cytoplasm, both BvIAA2 and BvIAA6 remained confined into the nucleus regardless of the presence of p25 suggesting their stabilization though p25 interaction. Overexpression of p25-interacting partners (BvIAA2, BvIAA6 and BvIAA28) in Nicotiana benthamiana induced an auxin-insensitive phenotype characterized by plant dwarfism and dramatically reduced LR development. Thus, our work reveals a distinct class of transcriptional repressors targeted by p25., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Muellender, Savenkov, Reichelt, Varrelmann and Liebe.)

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana . A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22 , ARF17/18 , and TIR , were down-regulated. GRF1 , which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus -induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin , and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana . These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

Beet necrotic yellow vein virus (BNYVV) is causal agent of rhizomania disease, which is the most devastating viral disease in sugar beet production leading to a dramatic reduction in beet yield and sugar content. The virus is transmitted by the ubiquitous distributed soil-borne plasmodiophoromycete Polymyxa betae that infects the root tissue of young sugar beet plants. Rz1 is the major resistance gene widely used in most sugar beet varieties to control BNYVV. The strong selection pressure on the virus population promoted the development of strains that can overcome Rz1 resistance. Resistance-breaking has been associated with mutations in the RNA3-encoded pathogenicity factor P25 at amino acid positions 67-70 (tetrad) as well as with the presence of an additional RNA component (RNA5). However, respective studies investigating the resistance-breaking mechanism by a reverse genetic system are rather scarce. Therefore, we studied Rz1 resistance-breaking in sugar beet using a recently developed infectious clone of BNYVV A-type. A vector free infection system for the inoculation of young sugar beet seedlings was established. This assay allowed a clear separation between a susceptible and a Rz1 resistant genotype by measuring the virus content in lateral roots at 52 dpi. However, mechanical inoculation of sugar beet leaves led to the occurrence of genotype independent local lesions, suggesting that Rz1 mediates a root specific resistance toward BNYVV that is not active in leaves. Mutation analysis demonstrated that different motifs within the P25 tetrad enable increased virus replication in roots of the resistant genotype. The resistance-breaking ability was further confirmed by the visualization of BNYVV in lateral roots and leaves using a fluorescent-labeled complementary DNA clone of RNA2. Apart from that, reassortment experiments evidenced that RNA5 enables Rz1 resistance-breaking independent of the P25 tetrad motif. Finally, we could identify a new resistance-breaking mutation, which was selected by high-throughput sequencing of a clonal virus population after one host passage in a resistant genotype. Our results demonstrate the feasibility of the reverse genetic system for resistance-breaking analysis and illustrates the genome plasticity of BNYVV allowing the virus to adapt rapidly to sugar beet resistance traits., (Copyright © 2020 Liebe, Wibberg, Maiss and Varrelmann.)

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Rhizomania of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is characterized by excessive lateral root (LR) formation leading to dramatic reduction of taproot weight and massive yield losses. LR formation represents a developmental process tightly controlled by auxin signaling through AUX/IAA-ARF responsive module and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcriptional network. Several LBD transcription factors play central roles in auxin-regulated LR development and act upstream of EXPANSINS (EXPs), cell wall (CW)-loosening proteins involved in plant development via disruption of the extracellular matrix for CW relaxation and expansion. Here, we present evidence that BNYVV hijacks these auxin-regulated pathways resulting in formation LR and root hairs (RH). We identified an AUX/IAA protein (BvAUX28) as interacting with P25, a viral virulence factor. Mutational analysis indicated that P25 interacts with domains I and II of BvAUX28. Subcellular localization of co-expressed P25 and BvAUX28 showed that P25 inhibits BvAUX28 nuclear localization. Moreover, root-specific LBDs and EXPs were greatly upregulated during rhizomania development. Based on these data, we present a model in which BNYVV P25 protein mimics action of auxin by removing BvAUX28 transcriptional repressor, leading to activation of LBDs and EXPs. Thus, the evidence highlights two pathways operating in parallel and leading to uncontrolled formation of LRs and RHs, the main manifestation of the rhizomania syndrome., (© 2018 BSPP and John Wiley & Sons Ltd.)

Two members of the Benyviridae family and genus Benyvirus, Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.