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The dahlia isolate of potato spindle tuber viroid (PSTVd) accumulates slowly and induces mild disease symptoms in tomato (Solanum lycopersicum, cv. Rutgers) plants in contrast to the intermediate isolate (PSTVd-I). The dahlia isolate (PSTVd-D) differs from PSTVd-I in eight locations: 42 and 43 in the terminal left (TL); 64/65, 311, and 312/313 in the pathogenicity (P); 118 and 126 in the variable (V); and 201 in the terminal right (TR) domains. To investigate the molecular determinants in the PSTVd-D genome responsible for the attenuation of symptom severity and lower replication/accumulation in tomato plants, a series of mutants between PSTVd-D and PSTVd-I were constructed by focusing first on the mutations in the TL and P domains in the left-hand half of the molecule. Then, more detailed analysis was performed on the three mutations at positions 118, 126, and 201 in the V and TR domains. One of these mutations is located around the boundary of the right border of the RY-motif, a predicted recognition site of Virp1, a viroid-binding protein. Of 14 mutants (seven based on PSTVd-D and the other seven based on PSTVd-I) examined, 11 propagated stably and three lost infectivity. Mutations in the TL and P domains (42U, 43C, 310U/C, and U or UU insertion to 311/312 in PSTVd mild types) majorly influenced the expression of mild-like symptoms. In contrast, when each of the mutations at 118, 126, and 201 in the V and TR domains were exchanged independently, they minimally influenced systemic accumulation and symptom expression. Mutants based on PSTVd-D with PSTVd-I-type mutations at nucleotide positions 118, 126, and/or 201 showed mild symptoms similar to PSTVd-D, but their systemic accumulation was a little faster than PSTVd-D. In contrast, mutants based on PSTVd-I with PSTVd-D-type mutations at 118, 126, and/or 201 nucleotide positions showed severe symptoms similar to PSTVd-I, and the systemic accumulation was similar to or a little slower than PSTVd-I. The nucleotide at position 201 could be changed to U, G, or A, but C was not acceptable for replication. Because introduction of C at the position 201 can change the loop structure at the right boundary of the RY-motif's consensus sequence, the loop structure may influence recognition by Virp1.