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

Laufer, Marlene; Mohammad, Hamza; Maiß, E.; Richert-Pöggeler, Katja; Dall’Ara, Mattia; Ratti, Claudio; Gilmer, David; Liebe, Sebastian; Varrelmann, Mark

doi:10.1016/j.virol.2018.01.029

Cited by 33 publications

(41 citation statements)

References 55 publications

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“…To determine whether the presence of BNYVV RNA3, which encodes the P25 virulence factor, is required for the activation of EXP and LBD genes (identified through the use of Rz1 and Rz2 resistant varieties as being important for rhizomania development), we performed infection experiments with recombinant viruses reassorted for RNA3 genomic segments. To this end, we took advantage of the availability of the infectious cDNA clones for BNYVV and Beet soil‐borne mosaic virus (BSBMV) (Laufer et al., ). As for BNYVV, BSBMV belongs to the genus Benyvirus , has a genome organization similar to BNYVV and infects sugar beet, but does not cause the ‘hairy root’ phenotype typical of rhizomania (Workneh et al., ).…”

Section: Resultsmentioning

confidence: 99%

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

Gil

Liebe²,

Thiel³

et al. 2018

Molecular Plant Pathology

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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.

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Section: Resultsmentioning

confidence: 99%

“…Reassortment experiments in B . vulgaris were conducted using infectious full‐length cDNA clones of BNYVV and BSBMV (Laufer et al., ). Different combinations of viral RNAs were first inoculated into B .…”

Section: Methodsmentioning

confidence: 99%

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

Gil

Liebe²,

Thiel³

et al. 2018

Molecular Plant Pathology

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“…Agrobacterium tumefaciens/ Agrobacterium fabrum) strain GV2260 according to Voinnet et al (1998) with an OD600 of 0.5. The inoculum consisted of BNYVV RNA1-3 cDNA clones mixed in equal amounts (Laufer et al, 2018b). Plants were kept for symptom development in a climate chamber at day and night temperatures of 24 and 18°C, respectively, and a 14 h photoperiod.…”

Section: Plant Materials and Virus Inoculationmentioning

confidence: 99%

“…PCR products were gel purified using the NucleoSpin Gel and PCR Clean-up system according to the manufactures instructions (Macherey-Nagel). Gibson assembly was applied as in vitro recombination method for the cloning of full-length cDNAs under control of 35S promoter from cauliflower mosaic virus into the linearized plasmid pDIVA according to Laufer et al (2018b). After assembly, in vitro recombination products were transformed into chemical competent Escherichia coli cells (strain DH5a) as described by Inoue et al (1990).…”

Section: Construction Of Infectious Rna3 and Rna5 Clonesmentioning

confidence: 99%

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

Liebe¹,

Wibberg

Maiß

et al. 2020

Front. Plant Sci.

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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.

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“…Full‐length infectious cDNA clones of BNYVV including B‐type isolate F‐13 and A‐type isolate Yu2 under the control of the T7 and CaMV 35S promoter have been constructed for reverse genetics analysis of BNYVV (Delbianco et al ., ; Laufer et al ., ). RNA3‐ and RNA5‐based replicons have been constructed for foreign gene expression in Beta macrocarpa , C. quinoa and N. benthamiana (Alice et al ., ; Delbianco et al ., ; Erhardt et al ., ; Laufer et al ., ; Schmidlin et al ., ). Recently, portions of the RNA2 read‐through domain (RTD) of BNYVV and Beet soil‐borne mosaic virus (BSBMV) were substituted by fluorescent reporter genes to investigate co‐ and superinfection of these two viruses in N. benthamiana (Laufer et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Jiang

Zhang

Liu

et al. 2019

Plant Biotechnology Journal

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

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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

Cited by 33 publications

References 55 publications

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

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

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

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

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