Intermolecular base-pairing interactions, a unique topology and exoribonuclease-resistant noncoding RNAs drive formation of viral chimeric RNAs in plants.

In plants, exoribonuclease-resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive. Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)-encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5'-end of RNA 2 and 3'-end of either RNA 3 or RNA 4 bearing conservative exoribonuclease-resistant 'coremin' region. Using computational tools and site-directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base-pairing interaction between 'coremin' and 3'-proximal part of the CP gene of RNA 2 as well as a stem-loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5'→3' exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs. Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base-pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5'→3' exoribonuclease conserved in all plant species.

The arms race between beet necrotic yellow vein virus and host resistance in sugar beet.

Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease in sugar beet (Beta vulgaris), which is controlled since more than two decades by cultivars harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favored by a high selection pressure on the soil-borne virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the RNA3 encoded pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse making investigations on the resistance-breaking mechanism rather difficult. Therefore, we applied a reverse genetic system for BNYVV (A type) to study Rz1 resistance-breaking by direct agroinoculation of sugar beet seedlings. The bioassay allowed a clear discrimination between susceptible and Rz1 resistant plants already four weeks after infection, and resistance-breaking was independent of the sugar beet Rz1 genotype. A comprehensive screen of natural tetrads for resistance-breaking revealed several new mutations allowing BNYVV to overcome Rz1. The supplementation of an additional RNA5 encoding the pathogenicity factor P26 allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. This suggests the presence of two distinct resistance-breaking mechanisms allowing BNYVV to overcome Rz1. Finally, we showed that the resistance-breaking effect of the tetrad and the RNA5 is specific to Rz1 and has no effect on the stability of the second resistance gene Rz2. Consequently, double resistant cultivars (Rz1+Rz2) should provide effective control of Rz1 resistance-breaking strains. Our study highlights the flexibility of the viral genome allowing BNYVV to overcome host resistance, which underlines the need for a continuous search for alternative resistance genes.

Comparative analysis of virus pathogenicity and resistance-breaking between the P- and A-type from the beet necrotic yellow vein virus using infectious cDNA clones.

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.

Manipulation of auxin signalling by plant viruses.

Compatible plant-virus interactions result in dramatic changes of the plant transcriptome and morphogenesis, and are often associated with rapid alterations in plant hormone homeostasis and signalling. Auxin controls many aspects of plant organogenesis, development, and growth; therefore, plants can rapidly perceive and respond to changes in the cellular auxin levels. Auxin signalling is a tightly controlled process and, hence, is highly vulnerable to changes in the mRNA and protein levels of its components. There are several core nuclear components of auxin signalling. In the nucleus, the interaction of auxin response factors (ARFs) and auxin/indole acetic acid (Aux/IAA) proteins is essential for the control of auxin-regulated pathways. Aux/IAA proteins are negative regulators, whereas ARFs are positive regulators of the auxin response. The interplay between both is essential for the transcriptional regulation of auxin-responsive genes, which primarily regulate developmental processes but also modulate the plant immune system. Recent studies suggest that plant viruses belonging to different families have developed various strategies to disrupt auxin signalling, namely by (a) changing the subcellular localization of Aux/IAAs, (b) preventing degradation of Aux/IAAs by stabilization, or (c) inhibiting the transcriptional activity of ARFs. These interactions perturb auxin signalling and experimental evidence from various studies highlights their importance for virus replication, systemic movement, interaction with vectors for efficient transmission, and symptom development. In this microreview, we summarize and discuss the current knowledge on the interaction of plant viruses with auxin signalling components of their hosts.

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1.

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.

The Virulence Factor p25 of Beet Necrotic Yellow Vein Virus Interacts With Multiple Aux/IAA Proteins From Beta vulgaris: Implications for Rhizomania Development.

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.

Comparative Transcriptome Analysis Provides Molecular Insights into the Interaction of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus with Their Host Sugar Beet.

Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) are closely related species, but disease development induced in their host sugar beet displays striking differences. Beet necrotic yellow vein virus induces excessive lateral root (LR) formation, whereas BSBMV-infected roots appear asymptomatic. A comparative transcriptome analysis was performed to elucidate transcriptomic changes associated with disease development. Many differentially expressed genes (DEGs) were specific either to BNYVV or BSBMV, although both viruses shared a high number of DEGs. Auxin biosynthesis pathways displayed a stronger activation by BNYVV compared to BSBMV-infected plants. Several genes regulated by auxin signalling and required for LR formation were exclusively altered by BNYVV. Both viruses reprogrammed the transcriptional network, but a large number of transcription factors involved in plant defence were upregulated in BNYVV-infected plants. A strong activation of pathogenesis-related proteins by both viruses suggests a salicylic acid or jasmonic acid mediated-defence response, but the data also indicate that both viruses counteract the SA-mediated defence. The ethylene signal transduction pathway was strongly downregulated which probably increases the susceptibility of sugar beet to Benyvirus infection. Our study provides a deeper insight into the interaction of BNYVV and BSBMV with the economically important crop sugar beet.

Application of a Reverse Genetic System for Beet Necrotic Yellow Vein Virus to Study Rz1 Resistance Response in Sugar Beet.

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.

Massive up-regulation of LBD transcription factors and EXPANSINs highlights the regulatory programs of rhizomania disease.

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.

Fluorescent labelling of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus for co- and superinfection experiments in Nicotiana benthamiana.

Infectious full-length clones of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), both genus Benyvirus, were used for fluorescent labelling with the objective to study their interaction in coinfection and superinfection experiments. Fluorescent labelling was achieved by replacing a part of the RNA2 encoded coat protein read-through domain with either GFP or mRFP fluorescent marker proteins. This resulted in a translational fusion comprising the coat and the fluorescent protein. The labelled viruses were infectious and moved systemically in Nicotiana benthamiana, producing wild-type-like symptoms. Virus particles could be observed by electron microscopy, demonstrating that the viral read-through domain is dispensable for particle formation. Coinfection experiments revealed a spatial separation of differentially labelled populations of both identical and different Benyvirus species after N. benthamiana agro-inoculation. Identical observations were obtained when Tobacco rattle virus (TRV) was differentially labelled and used for coinfection. In contrast, coinfections of BSBMV with Potato virus X (PVX) or TRV resulted in many co-infected cells lacking spatial separation. Micro-projectile co-bombardment of N. benthamiana leaves revealed that two differently labelled populations of the same virus co-infected only a few cells before starting to separate. In superinfection experiments with N. benthamiana, BSBMV and BNYVV were unable to establish a secondary infection in plants that were previously infected with BNYVV or BSBMV. Taken together, this is the first work to describe the interaction between two economically important Benyviruses using fluorescence-labelled full-length clones.

Biological properties of Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones produced by isothermal in vitro recombination: Insights for reassortant appearance.

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.

Taxonomic analysis of the microbial community in stored sugar beets using high-throughput sequencing of different marker genes.

Post-harvest colonization of sugar beets accompanied by rot development is a serious problem due to sugar losses and negative impact on processing quality. Studies on the microbial community associated with rot development and factors shaping their structure are missing. Therefore, high-throughput sequencing was applied to describe the influence of environment, plant genotype and storage temperature (8°C and 20°C) on three different communities in stored sugar beets, namely fungi (internal transcribed spacers 1 and 2), Fusarium spp. (elongation factor-1α gene fragment) and oomycetes (internal transcribed spacers 1). The composition of the fungal community changed during storage mostly influenced by the storage temperature followed by a weak environmental effect. Botrytis cinerea was the prevalent species at 8°C whereas members of the fungal genera Fusarium and Penicillium became dominant at 20°C. This shift was independent of the plant genotype. Species richness within the genus Fusarium also increased during storage at both temperatures whereas the oomycetes community did not change. Moreover, oomycetes species were absent after storage at 20°C. The results of the present study clearly show that rot development during sugar beet storage is associated with pathogens well known as causal agents of post-harvest diseases in many other crops.

Effect of Environment and Sugar Beet Genotype on Root Rot Development and Pathogen Profile During Storage.

Storage rots represent an economically important factor impairing the storability of sugar beet by increasing sucrose losses and invert sugar content. Understanding the development of disease management strategies, knowledge about major storage pathogens, and factors influencing their occurrence is crucial. In comprehensive storage trials conducted under controlled conditions, the effects of environment and genotype on rot development and associated quality changes were investigated. Prevalent species involved in rot development were identified by a newly developed microarray. The strongest effect on rot development was assigned to environment factors followed by genotypic effects. Despite large variation in rot severity (sample range 0 to 84%), the spectrum of microorganisms colonizing sugar beet remained fairly constant across all treatments with dominant species belonging to the fungal genera Botrytis, Fusarium, and Penicillium. The intensity of microbial tissue necrotization was strongly correlated with sucrose losses (R² = 0.79 to 0.91) and invert sugar accumulation (R² = 0.91 to 0.95). A storage rot resistance bioassay was developed that could successfully reproduce the genotype ranking observed in storage trials. Quantification of fungal biomass indicates that genetic resistance is based on a quantitative mechanism. Further work is required to understand the large environmental influence on rot development in sugar beet.

Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present.