Barley stripe mosaic virus North Dakota 18 (ND18), Beijing (BJ), Xinjiang (Xi), Type (TY) and CV21 strains are unable to infect the Brachypodium distachyon Bd3-1 inbred line, which harbours a resistance gene designated Bsr1, but the Norwich (NW) strain is virulent on Bd3-1. Analysis of ND18 and NW genomic RNA reassortants and RNA beta mutants demonstrates that two amino acids within the helicase motif of the triple gene block 1 (TGB1) movement protein have major effects on their Bd3-1 phenotypes. Resistance to ND18 correlates with an arginine residue at TGB1 position 390 (R-390) and a threonine at position 392 (T-392), whereas the virulent NW strain contains lysines (K) at both positions. ND18 TGB1 R390K ((ND)TGB1(R390K)) and (ND)TGB1(T392K) single substitutions, and an (ND)TGB1(R390K,T392K) double mutation resulted in systemic infections of Bd3-1. Reciprocal (ND)TGB1 substitutions into (NW)TGB1 ((NW)TGB1(K390R) and (NW)TGB1(K392T)) failed to affect virulence, implying that K-390 and K-392 compensate for each other. In contrast, an (NW)TGB1(K390R,K392T) double mutant exhibited limited vascular movement in Bd3-1, but developed prominent necrotic streaks that spread from secondary leaf veins. This phenotype, combined with the appearance of necrotic spots in certain ND18 mutants, and necrosis and rapid wilting of Bd3-1 plants after BJ strain ((BJ)TGB1(K390,T392)) inoculations, show that Bd3-1 Bsr1 resistance is elicited by the TGB1 protein and suggest that it involves a hypersensitive response.