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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]