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Effect of sugar beet genotype on the Beet necrotic yellow vein virus?P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains.

ABSTRACT: Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67-70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep sequencing. The level of P25 variation in the populations analysed did not correlate with RB properties. Remarkably, one WT population contained P25 with RB mutations at a frequency of 11%. To demonstrate selection by Rz1 and the influence of RB mutations on relative fitness, competition experiments between strains were performed. Following a mixture of strains with four RNAs, a shift in tetrad variants was observed, suggesting that strains did not mix or transreplicate. The plant genotype exerted a clear influence on the frequency of RB tetrads. In Rz1 plants, the RB variants outcompeted the WT variants, and mostly vice versa in susceptible plants, demonstrating a relative fitness penalty of RB mutations. The strong genotype effect supports the hypothesized Rz1?RB strain selection with four RNAs, suggesting that a certain tetrad needs to become dominant in a population to influence its properties. Tetrad selection was not observed when an RB strain, with an additional P26 protein encoded by a fifth RNA, competed with a WT strain, supporting its role as a second BNYVV pathogenicity factor and suggesting the reassortment of both types.

SUBMITTER: Bornemann K

PROVIDER: S-EPMC6638868 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains.

Bornemann Kathrin K Varrelmann Mark M

Molecular plant pathology 20130103 4

Beet necrotic yellow vein virus (BNYVV), vectored by Polymyxa betae, causes rhizomania in sugar beet. For disease control, the cultivation of hybrids carrying Rz1 resistance is crucial, but is compromised by resistance-breaking (RB) strains with specific mutations in the P25 protein at amino acids 67-70 (tetrad). To obtain evidence for P25 variability from soil-borne populations, where the virus persists for decades, populations with wild-type (WT) and RB properties were analysed by P25 deep seq ...[more]

PMID: 23282068

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Project description: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.

Project description:Title : Characterization of genes differentially expressed in roots of transgenic arabidopsis lines expressing the p25 protein of beet necrotic yellow vein virus. Biological question : Rhizomania ("crazy root") is a severe disease of sugar beet caused by beet necrotic yellow vein virus (BNYVV), which is transmitted by the soil-inhabiting fungus Polymyxa betae. Symptoms of virus infection are characterized by a constricted tap root and a massive proliferation of fine rootlets that often undergo necrosis. BNYVV RNA-3 encodes a 25 kDa (p25) which is an important determinant of leaf symptom phenotype. It also governs BNYVV invasion of the plant root system and induction of rootlet proliferation in sugar beet. In order to obtain a better understanding of molecular aspects of disease development in roots and to characterize specific host genes involved in response to viral infection, transgenic Arabidopsis overexpressors of p25 viral protein was obtained and better characterized. It was shown that transgenic plants that efficiently expressed p25 protein produced more lateral roots. Comparative analysis (microarray) was performed between wild type Arabidopsis roots and transgenic Arabidopsis roots expressing p25 protein, in order to identify Arabidopsis genes differentially expressed in response to p25 viral protein. Experiment description: Seeds were surface sterilized, chilled at 4C for 4 days, and then germinated and grown on square Petri plates containing sterilized Murashige and Skoog (MS) medium with 1% sucrose. Such stock plates were arranged vertically in plastic racks and placed into growth chamber. After 5 days, plants were transferred carefully onto fresh MS medium big round plates. On each plate, 60 Wild Type (WT) plantlets were transferred on the half right of the plate, and 60 transgenic plantlets (B, E or T lines) were transferred on the half left of the plate. Plates were arranged horizontally and placed into growth chamber. Experiment 1 : 5 plates containing WT0A control plants and B0A transgenic plants. Experiment 2 : 5 plates containing WT1 control plants and B transgenic plants. 5 plates containing WT2 control plants and E transgenic plants. 5 plates containing WT3 control plants and T transgenic plants. Plants were harvested after 7 days (experiment 1) or 12 days (experiment 2), and WT roots or transgenic roots were pooled and conserved at -80C.

Project description:Herein, proteomic profiling was conducted on susceptible sugarbeet infected with BNYVV to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugarbeet leaf tissue infected with BNYVV (beet necrotic yellow vein virus), quantified, and analyzed by mass spectrometry. A proprietary sugarbeet variety, KWS-rzNI, susceptible to all forms of BNYVV, was provided by KWS (Einbeck, Germany). To enable detection of a broad range of proteins associated with compatible interactions, two sources of BNYVV were used; standard BNYVV pathotype A, originally collected from Spence Field at the USDA-ARS in Salinas, CA, and resistance-breaking BNYVV-IV originating from the field, Rockwood 158, Imperial County, CA. Pooled leaf tissue from each cup was ground in liquid nitrogen and lyophilized. Total protein was extracted from 100 mg (dry weight) of lyophilized leaf tissue of each sample using the Plant Total Protein Extraction Kit (Sigma, St. Louis, MO) according to manufacturer’s recommendations. Protease inhibitors were added to liquid protein extracts (Pierce, Rockford, IL), and samples were stored at -80oC prior to analysis. Protein concentrations were determined via Bradford Assay [10] (Thermo Scientific, Rockford, IL) and 30 g of each sample underwent in-solution proteolytic digestion as previously described [11]. Briefly, samples were solubilized in 8 M urea, 0.2% Protease Max (Promega, Madision, WI), then reduced with dithiothreitol, alkylated with iodoacetamide, and digested with 1% Protease Max and trypsin at 37oC for 3 hours. Samples were dried in a Speed Vac® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ull 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). 1 l of each sample (three replicates from each soil type) was analyzed in duplicate injections via LC-MS/MS . Peptides were purified and concentrated using an on-line enrichment column (Thermo Scientific 5m, 100 m ID x 2 cm C18 columns). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Thermo Scientific EASYnano-LC, 3m, 75 m ID x 100 mm C18 column) using a 90 minute linear gradient from 10%-30% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos Pro) and spectra collected over a m/z range of 400-2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group. MS/MS spectra were searched against a Uniprot Amaranthacae database (3,645 entries, version 03/06/13) concatenated to a reverse database and the Uniprot Mus musculus database (50,665 entries, version 03/06/13) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). Due to the limited size of the Amaranthaceae database, the Mus musculus sequences were added to ensure adequate database size for statistical scoring and calculation of false discovery rates. The following search parameters were used in Mascot: monoisotopic mass, parent mass tolerance of 20 ppm, fragment ion mass tolerance of 0.8 Da , complete tryptic digestion allowing two missed cleavages, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. SEQUEST search parameters were the same except for a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 0.0120 Da. Peptide identifications from both of the search engines were combined using probabilistic protein identification algorithms in Scaffold 4 [13, 14] (Version 4.0.3, Proteome Software, Portland, OR) with protein clustering enabled. All data files were then combined using the “mudpit” option in Scaffold 4 generating a composite listing for all proteins identified. Thresholds were set to 99% protein probability, 95% peptide probability, and a 2 unique peptide minimum was required. The peptide FDR was 0% after manual validation of all proteins identified by 2 unique peptides [15]. Criteria for manual validation included the following: 1) a minimum of at least 5 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.

Project description:Rhizomania disease, caused by the Beet necrotic yellow vein virus (BNYVV), is considered as one of the major constraints for sugar beet production, worldwide. As a result of the introgression of major resistance genes (Holly, Rz2) in commercially available sugar beet varieties, the virus has endured strong selection pressure since the 90s'. Understanding the virus response and diversity to sugar beet resistance is a key factor for a sustainable management of only few resistance genes. Here we report rhizomania surveys conducted in a rhizomania hot spot, the Pithiviers area (France) during a 4-year period and complementary to the study of Schirmer et al. (2005). The study aimed at evaluating the intra- and inter-field BNYVV diversity in response to different sources of resistance and over the growing season. To follow rhizomania development over the sugar beet growing season, extensive field samplings combined with field assays were performed in this study. The evolution of the BNYVV diversity was assessed at intra- and inter-field levels, with sugar beet cultivars containing different resistance genes (Rz1, Rz1 + Heterodera schachtii resistance and Rz1Rz2). Intra-field diversity was analyzed at the beginning and the end of the growing season of each field. From more than one thousand field samples, the simultaneous presence of the different A, B and P types of BNYVV was confirmed, with 21 variants identified at positions 67-70 of the p25 tetrad. The first variant, AYHR, was found most commonly followed by SYHG. Numerous mixed infections (9.93% of the samples), mostly of B-type with P-type, have also been evidenced. Different tetrads associated with the A- or B-type were also found with a fifth RNA-genome component known to allow more aggressiveness to BNYVV on sugar beet roots. Cultivars with Rz1+Rz2 resistant genes showed few root symptoms even if the BNYVV titre was quite high according to the BNYVV type present. The virus infectious potential in the soil at the end of the growing season with such cultivars was also lower despite a wider diversity at the BNYVV RNA3 sequence level. Rz1+Rz2 cultivars also exhibited a lower presence of Beet soil-borne virus (BSBV), a P. betae-transmitted Pomovirus. Cultivars with Rz1 and nematode (N) resistance genes cultivated in field infected with nematodes showed lower BNYVV titre than those with Rz1 or Rz1+Rz2 cultivars. Overall, the population structure of BNYVV in France is shown to be different from that previously evidenced in different world areas. Implications for long-term management of the resistance to rhizomania is discussed.

Dataset Information

Effect of sugar beet genotype on the Beet necrotic yellow vein virus?P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains.

Publications

Effect of sugar beet genotype on the Beet necrotic yellow vein virus P25 pathogenicity factor and evidence for a fitness penalty in resistance-breaking strains.

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