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.

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.

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.

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.

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.