Rapid and specific detection of Pentastiridius leporinus by recombinase polymerase amplification assay.

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of an emerging and fast spreading sugar beet disease, the syndrome 'basses richesses' (SBR), in different European countries. The disease is caused by the γ-3-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the phytoplasma 'Candidatus Phytoplasma solani' which are exclusively transmitted by planthoppers and can lead to a significant loss of sugar content and yield. Monitoring of this insect vector is important for disease management. However, the morphological identification is time consuming and challenging as two additional cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with a very close morphology have been reported in sugar beet fields. Further, identification of females and nymphs of P. leporinus at species level based on taxonomic key is not possible. In this study, an isothermal nucleic acid amplification based on recombinase polymerase amplification (RPA) was developed to specifically detect P. leporinus. In addition, real-time RPA was developed to detect both adults (male and female) and nymph stages using pure or crude nucleic acid extracts. The sensitivity of the real-time RPA for detection of P. leporinus was comparable to real-time PCR, but a shorter time (< 7 min) was required. This is a first report for real-time RPA application for P. leporinus detection using crude nucleic acid templates which can be applied for fast and specific detection of this vector in the field.

Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

Effects of succession crops and soil tillage on suppressing the syndrome 'basses richesses' vector Pentastiridius leporinus in sugar beet.

BACKGROUND: Pentastiridius leporinus (Hemiptera: Cixiidae) is the most important vector of syndrome 'basses richesses' (SBR), a new disease that leads to severe economic losses in sugar beet. In this study, different soil tillage methods (ploughing and cultivator) and crops (winter wheat, spring wheat, maize and bare soil) following SBR-infested sugar beet were tested as potential management options in field trials. In the laboratory, the survival and development of first and third instar nymphs on wheat and maize was studied to further assess their suitability as host plants. RESULTS: In five out of seven field sites, reduced soil tillage had no effect on adult planthopper emergence compared to ploughing. In two sites, reduced tillage resulted in higher emergence rates. In nearly all field sites, up to 98.9% fewer emerging adults were detected in bare soil and maize, when compared to winter wheat. Under laboratory conditions, the lowest survival rate was found in first instar nymphs feeding on maize seedlings (4.2%), while 66.7% survived on wheat, over a period of 300 days. In contrast, 73.3% and 70% of third instar nymphs survived on wheat and maize over a period of 150 days. CONCLUSION: Soil tillage had little effect against Pentastiridius leporinus. Maize is a poor host for first instars but a suitable resource for third instar nymphs, the stage which encounters maize under field conditions. Hence, reductions in planthopper emergence in the field were likely caused by starvation due to the long host-free period between sugar beet harvest and the sowing of maize. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Beet mosaic virus expression of a betalain transcription factor allows visual virus tracking in Beta vulgaris.

In the field of plant virology, the usage of reverse genetic systems has been reported for multiple purposes. One is understanding virus-host interaction by labelling viral cDNA clones with fluorescent protein genes to allow visual virus tracking throughout a plant, albeit this visualization depends on technical devices. Here we report the first construction of an infectious cDNA full-length clone of beet mosaic virus (BtMV) that can be efficiently used for Agrobacterium-mediated leaf inoculation with high infection rate in Beta vulgaris, being indistinguishable from the natural virus isolate regarding symptom development and vector transmission. Furthermore, the BtMV clone was tagged with the genes for the monomeric red fluorescent protein or the Beta vulgaris BvMYB1 transcription factor, which activates the betalain biosynthesis pathway. The heterologous expression of BvMYB1 results in activation of betalain biosynthesis genes in planta, allowing visualization of the systemic BtMV spread with the naked eye as red pigmentation emerging throughout beet leaves. In the case of BtMV, the BvMYB1 marker system is stable over multiple mechanical host passages, allows qualitative as well as quantitative virus detection and offers an excellent opportunity to label viruses in plants of the order Caryophyllales, allowing an in-depth investigation of virus-host interactions on the whole plant level.

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.

Disease Incidence and Severity of Cercospora Leaf Spot in Sugar Beet Assessed by Multispectral Unmanned Aerial Images and Machine Learning.

Disease incidence (DI) and metrics of disease severity are relevant parameters for decision making in plant protection and plant breeding. To develop automated and sensor-based routines, a sugar beet variety trial was inoculated with Cercospora beticola and monitored with a multispectral camera system mounted to an unmanned aerial vehicle (UAV) over the vegetation period. A pipeline based on machine learning methods was established for image data analysis and extraction of disease-relevant parameters. Features based on the digital surface model, vegetation indices, shadow condition, and image resolution improved classification performance in comparison with using single multispectral channels in 12 and 6% of diseased and soil regions, respectively. With a postprocessing step, area-related parameters were computed after classification. Results of this pipeline also included extraction of DI and disease severity (DS) from UAV data. The calculated area under disease progress curve of DS was 2,810.4 to 7,058.8%.days for human visual scoring and 1,400.5 to 4,343.2%.days for UAV-based scoring. Moreover, a sharper differentiation of varieties compared with visual scoring was observed in area-related parameters such as area of complete foliage (AF), area of healthy foliage (AH), and mean area of lesion by unit of foliage ([Formula: see text]). These advantages provide the option to replace the laborious work of visual disease assessments in the field with a more precise, nondestructive assessment via multispectral data acquired by UAV flights.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular Detection of Pentastiridius leporinus, the Main Vector of the Syndrome 'Basses Richesses' in Sugar Beet.

Monitoring of Pentastiridius leporinus (Hemiptera: Auchenorrhyncha: Cixiidae), representing the main vector of the syndrome 'basses richesses' (SBR) disease in sugar beet is based on morphological identification. However, two other cixiid species, Reptalus quinquecostatus and Hyalesthes obsoletus with similar external characters are known to appear in sugar beet fields and are challenging to be distinguished from P. leporinus. We present a PCR-based method for species-specific detection of both male and female P. leporinus, directly after sweep net collection or after up to 18 months long term storage on sticky traps. Two methods of DNA template preparation, based on a commercial extraction kit or on simple grinding in phosphate-buffered saline (PBS) were compared. The latter method was also established for eggs and all five nymphal instars of P. leporinus from a rearing. Furthermore, in silico primer analysis showed that all Auchenorrhyncha species including far related species reported from sugar beet fields can be differentiated from P. leporinus. This was PCR-confirmed for the most common Auchenorrhyncha species from different German sugar beet fields. Sequence analysis of the P. leporinus mitochondrial cytochrome oxidase I gene (COI) amplicon showed a close relationship to COI from P. beieri but separated from the Reptalus and Hyalesthes species which are grouped into the same family Cixiidae. We present a sensitive, cost- and time-saving PCR-based method for reliable and specific detection of eggs and all nymphal instars, as well as male and female P. leporinus, after different methods of planthopper collection and template DNA template preparation that can be used in large scale monitoring assays.

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 Rep and C1 of Beet curly top Iran virus represent pathogenicity factors and induce hypersensitive response in Nicotiana benthamiana plants.

Beet curly top Iran virus (BCTIV) is a member of the genus Becurtovirus (Family Geminiviridae) with a circular single-strand DNA genome. BCTIV causes leaf curling and vein swelling symptoms in plants. However, the potential pathogenicity factor/s in BCTIV is/are not known. This study presents characterization of complementary-sense transcripts of BCTIV and the viral factors in directing the pathogenicity and hypersensitive response (HR) in Nicotiana benthamiana plants. In both local and systemic infection, splicing of the complementary transcripts of BCTIV was observed. Notably, a small number (8.3%) of transcripts were spliced to produce Rep (C1:C2) transcripts after deletion of 155 nt (position 1892-2046 from BCTIV). Expression of BCTIV genes in N. benthamiana using tobacco rattle virus (TRV)-based vector showed that Rep together with C1 are the main pathogenicity factors which cause typical viral leaf curling symptoms. In addition, the V2 caused a mild leaf curling, thickening, and asymmetric leaves, while the V1, V3, and C2 had no clear effect on the plant phenotype. Transient expression of individual viral genes showed that both the C1 and Rep trigger a HR response in N. benthamiana. The higher expression of HR marker genes, harpin-induced 1 (Hin1) and hypersensitivity-related (Hsr203JI), supported the role of C1 and Rep in HR response in plants. It is concluded that Rep and C1 are the main pathogenicity factors that also trigger HR response in plants.

Life history traits and a method for continuous mass rearing of the planthopper Pentastiridius leporinus, a vector of the causal agent of syndrome 'basses richesses' in sugar beet.

BACKGROUND: The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of the γ-3 proteobacterium 'Candidatus Arsenophonus phytopathogenicus' which causes the syndrome 'basses richesses' (SBR) in sugar beet. SBR is a new and fast-spreading disease in Central Europe that leads to high yield losses. To date, the development of management strategies has been hampered by insufficient knowledge about general life history traits of the planthopper and, most importantly, the year-round availability of insects reared under controlled conditions. Rearing of P. leporinus has been considered challenging and to date no protocol exists. RESULTS: Here we describe a method for mass rearing P. leporinus on sugar beet from egg to adult that has produced five generations and over 20 000 individuals between June 2020 and March 2022. An alternative host such as wheat is not necessary for completing the life cycle. No-choice experiments showed that P. leporinus lays 139.1 ± 132.9 eggs on sugar beet, whereas no oviposition was observed on its nymphal host wheat. Head capsule width was identified as a trait that unequivocally distinguished the five nymphal instars. Developmental time from first instar to adult was 193.6 ± 35.8 days for males and 193.5 ± 59.2 days for females. Infection rates of adults were tested with a nested polymerase chain reaction. The results demonstrated that 70%-80% of reared planthoppers across all generations carried the SBR proteobacterium. CONCLUSION: The mass-rearing protocol and life history data will help overcome an important bottleneck in SBR research and enhance efforts in developing integrated pest management tools. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components.

Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.

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.

Evidence for the association of target-site resistance in cyp51 with reduced DMI sensitivity in European Cercospora beticola field isolates.

BACKGROUND: Cercospora leaf spot caused by Cercospora beticola is the most relevant foliar disease in sugar beet cultivation. In the last decade a decreasing sensitivity of C. beticola towards demethylation inhibitors (DMIs) occurred. Different mechanisms mediating a reduced sensitivity towards DMIs have been identified in different plant pathogens to date, such as target site mutations, over-expression or active excretion of the fungicide. RESULTS: A sequencing of the cytochrome P450-dependent sterol 14α-demethylase gene sequence (cyp51) of diverse C. beticola isolates collected in different European countries with reduced DMI sensitivity was performed in order to find a possible correlation of mutations with higher EC50 values. The amino acid alterations Y464S, L144F and I309T combined with L144F were found to be associated with a reduced sensitivity. Furthermore, mutations I387M, M145W and M145W with E460Q were found uniquely. Additionally, constitutive and fungicide triggered expression of cyp51 was assayed by means of RT-qPCR. A very strong induction of cyp51 mRNA expression in sensitive isolates suggests that the fungal cells upregulate expression to maintain ergosterol biosynthesis in DMI presence. The less intensive cyp51 induction in isolates with higher EC50 values underlines the possible correlation of the found target-site mutations with reduced sensitivity. CONCLUSION: This study provides new results about possible alterations in the target gene mediating reduced sensitivity of C. beticola towards DMIs and hypothesized a fungicide induced over-expression of the target enzyme CYP51 as natural reaction of the fungus to fungicide application. © 2020 Society of Chemical Industry.

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.

Production of a Beet chlorosis virus full-length cDNA clone by means of Gibson assembly and analysis of biological properties.

Beet chlorosis virus (genus Polerovirus, family Luteoviridae), which is persistently transmitted by the aphid Myzus persicae, is part of virus yellows in sugar beet and causes interveinal yellowing as well as significant yield loss in Beta vulgaris. To allow reverse genetic studies and replace vector transmission, an infectious cDNA clone under cauliflower mosaic virus 35S control in a binary vector for agrobacterium-mediated infection was constructed using Gibson assembly. Following agroinoculation, the BChV full-length clone was able to induce a systemic infection of the cultivated B. vulgaris. The engineered virus was successfully aphid-transmitted when acquired from infected B. vulgaris and displayed the same host plant spectrum as wild-type virus. This new polerovirus infectious clone is a valuable tool to identify the viral determinants involved in host range and study BChV protein function, and can be used to screen sugar beet for BChV resistance.

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.