The beet necrotic yellow vein virus (BNYVV) causes the rhizomania disease in sugar beet, which is controlled since more than two decades by varieties harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favoured by a high selection pressure on the virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse with more than 25 known tetrad variants and different RNA5 species making investigations on the resistance-breaking mechanism rather difficult. Therefore, we refined our previously developed reverse genetic system for BNYVV (A type) to study the Rz1 resistance-breaking mechanism by direct agroinoculation of sugar beet seedlings. Our system allowed a clear discrimination between susceptible and resistant genotypes. A subsequent screen for resistance-breaking revealed that multiple tetrad variants allow BNYVV to overcome Rz1. Furthermore, certain mutations severely impaired the viral pathogenicity in the susceptible genotype suggesting a crucial function of the tetrad in P25. Finally, the supplementation of an additional RNA5 species, either from the J or P type group, allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. However, this effect was impaired in a genotype carrying Rz2, which is another resistance gene against BNYVV. Our infection system enables a rapid identification resistance-breaking mutations and highlights the plasticity of the BNYVV genome allowing adaption towards Rz1.