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.

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.

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.

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.

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.

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.

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.

Identification of a novel mycovirus isolated from Rhizoctonia solani (AG 2-2 IV) provides further information about genome plasticity within the order Tymovirales.

The complete genome of a novel mycovirus, named Rhizoctonia solani flexivirus 1 (RsFV-1), which infects an avirulent strain of Rhizoctonia solani AG 2-2 IV, was sequenced and analyzed. Its RNA genome consists of 10,621 nucleotides, excluding the poly-A tail, and encodes a single protein of 3477 amino acids. The identification of conserved motifs of methyltransferase, helicase and RNA-dependent RNA polymerase revealed its relatedness to members of the alphavirus-like superfamily of positive-strand RNA viruses. Phylogenetic analysis of these fused domains suggested that this virus should be assigned to the order Tymovirales. The recently described Fusarium graminearum deltaflexivirus 1 was found to be its closest relative. However, the whole genome, as well as the encoded protein of RsFV-1, is larger than that of other known members of the order Tymovirales, and unlike all other viruses belonging to this order, its methyltransferase domain is not located at the N-terminus of the replicase. Although genome diversity, as a result of recombination and gene loss, is a well-documented trait in members of the order Tymovirales, no related virus with a comparable genome alteration has been reported before. For these reasons, RsFV-1 broadens our perception about genome plasticity and diversity within the order Tymovirales.

Control of Rhizoctonia solani in Sugar Beet and Effect of Fungicide Application and Plant Cultivar on Inoculum Potential in the Soil.

Rhizoctonia solani (AG 2-2 IIIB) is the causal agent of Rhizoctonia root and crown rot, a disease that causes severe economic problems in sugar beet growing areas worldwide. In the United States, azoxystrobin is the most important active ingredient for fungicidal control of R. solani in sugar beet, showing efficacy superior to other substances. First reports on resistance development in R. solani, however, underline the importance of a careful fungicide resistance management. For this reason, the efficacy of a new fungicide mixture of azoxystrobin and difenoconazole was compared with a fungicide containing only azoxystrobin. Field trials were carried out under natural infection conditions as well as with inoculation in the years 2012, 2013, and 2014. Evaluation of the disease severity and the obtained white sugar yield of different sugar beet cultivars demonstrated that both fungicide treatments possess a similar efficacy, reducing the diseased beet surface by up to 78% and preventing yield losses. Additionally, a real-time PCR assay, based on DNA extracts from representative soil samples (250 g), was used to directly determine the effect of chemical treatment and plant cultivar on the soil-borne inoculum. Fungicide application significantly reduced the concentration of soil-borne inoculum by up to 97%. Furthermore, the results demonstrated that the cultivation of a susceptible cultivar significantly increases the concentration of R. solani in the soil by a factor of 200. In conclusion, the study implies that only a combination of resistant cultivar and fungicide application can prevent an accumulation of R. solani inoculum under conducive conditions in infested fields.

Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes.

BACKGROUND: Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. RESULTS: The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. CONCLUSIONS: The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology.

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.

Effect of Sugar Beet Variety and Nonhost Plant on Rhizoctonia solani AG2-2IIIB Soil Inoculum Potential Measured in Soil DNA Extracts.

A direct soil DNA extraction method from soil samples (250 g) was applied for detection of the soilborne sugar-beet-infecting pathogen Rhizoctonia solani anastomosis group (AG) 2-2IIIB using a newly developed real-time polymerase chain reaction assay that showed specificity to AG2-2IIIB when tested against various R. solani AG. The assay showed a good relation between cycle threshold and amount of AG2-2IIIB sclerotia detected in three spiked field soils and was also able to detect the pathogen in naturally infested field soil samples. A field trial was conducted to quantify R. solani AG2-2IIIB soil inoculum potential (IP) before and after growing a susceptible and a resistant sugar beet variety as well as after subsequent growth of an expected nonhost winter rye. Plants of the susceptible sugar beet variety displayed a higher disease severity. A more than sixfold increase of the R. solani AG2-2IIIB soil IP was observed in contrast to the resistant variety that resulted in a constant IP. Growing winter rye significantly reduced soil IP to the initial level at sowing. Further research is required to better understand the interaction between disease occurrence and soil IP as well as the environmental influence on IP development.

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.

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.

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 P25 pathogenicity factor of Beet necrotic yellow vein virus targets the sugar beet 26S proteasome involved in the induction of a hypersensitive resistance response via interaction with an F-box protein.

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.

Three Distinct Nanoviruses, One of Which Represents a New Species, Infect Faba Bean in Ethiopia.

In all, 70 of 296 leaf samples (23.6%) collected from faba bean (Vicia faba) plants showing leaf yellowing and stunting in Ethiopia gave nanovirus-positive reactions when studied by triple-antibody sandwich enzyme-linked immunosorbent assay using broad-spectrum monoclonal antibodies (MAbs) specific to nanoviruses. Further analysis of these samples with seven discriminating MAbs revealed contrasting epitope profiles that were categorized into roughly three serogroups, designated A, B, and C. Serogroup A was found in 89% of the nanovirus-positive samples whereas serogroups B and C were infrequently encountered. Sequence analysis of DNA-S and DNA-U1 of serogroup A, B, and C isolates suggested that each represents a distinct nanovirus species. Serogroup A comprised isolates of Faba bean necrotic stunt virus reported earlier only from Ethiopia and Morocco. The DNA-R, -S, -U1, and -U2 sequences of a serogroup B isolate closely resembled those of Faba bean necrotic yellows virus, providing first molecular evidence for its occurrence in Ethiopia. Sequence analysis of the eight genomic DNAs of a representative serogroup C isolate (Eth-231) showed that it shared overall nucleotide and amino acid sequence identities of only ≤70 and ≤74%, respectively, with other nanoviruses. This suggests that Eth-231 represents a new nanovirus species, for which the name faba bean yellow leaf virus is proposed.

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.