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

Abstract

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.

Pathogenetic roles of beet necrotic yellow vein virus RNA5 in the exacerbation of symptoms and yield reduction, development of scab‐like symptoms, and Rz1‐resistance breaking in sugar beet

“…Galein et al (2018) examined the variability and pathogenicity of BNYVV isolates from an area containing A-, B-and P-types, and found that P-type virus (with RNA5) showed the highest aggressiveness, although there is the complexity of genome reassortments between different types of BNYVV strains. Furthermore, Liebe et al (2020) showed that the presence of P-type RNA5 in the background of the A-type virus clone increased virus accumulation in the resistant plants, suggesting that this P-type RNA5 is involved in Rz1-RB. The present study here has provided more definite evidence on the effect of RNA3 and RNA5 on virus accumulation levels in Rz1-RB.…”

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses

“…The nucleotides at this location are considered to be among the most hypervariable within all eukaryotic viruses [ 25 , 34 , 43 ], a feature observed in our own recent study [ 17 ]. Moreover, recent associative [ 16 , 17 , 34 ] and functional [ 5 , 44 ] evidence suggests that variability in this region may account for the ability for some strains of the virus to circumvent dominant resistance genes in the sugar beet crop. As additional cases exist where a specific tetrad has been observed in both Rz-gene controlled and Rz-gene breaking isolates (e.g., tetrad ACHG [ 17 ]), it is possible that other changes in the genome may operate in conjunction with mutations in the tetrad motif or independent of this element in compromising host resistance.…”

“…The two largest RNAs of the BNYVV genome, RNAs 1 and 2, collectively represent over 70% of the virus genome and encode the replication, packaging, cell–cell movement, and silencing suppression functions of the virus. In contrast to most other biologically-active clones of BNYVV that provide infection through transient genome transcription consequent to Agrobacterium infiltration (i.e., “agro-infection”; [ 44 , 45 , 46 ]), we chose to employ in vitro production of capped RNA transcriptions as the means to produce inoculum for the infection in recipient cells in a manner more consistent with that found in nature. Along with previous reports of others who used NGS data in the construction of clones from which infectious RNA was produced either through in vitro transcription or via agro-infection [ 47 ], our study validates this approach in constructing clones for the study of BNYVV.…”

Pest categorisation of beet necrotic yellow vein virus

“…Severe disease symptoms are the reduced size of the roots and typical constrictions (funnel or ‘wineglass’‐like shape) of the infected taproots sometimes with nodules (small tumorous growths) along the taproot. Necrosis of vascular tissues (Figure 1) (Liebe et al., 2020) starting with a pale yellow to dark brown discoloration and necrotic rings may also occur. In less heavily infested plants, symptoms may affect only one lateral root of the plant (McGrann et al., 2009; Rush et al., 2006; Neher and Gallian, 2014; EPPO, 2006).…”