P-589 - DNA secondary structures formed by iteron motifs of a geminivirus regulate viral replication and systemic spread

Tomato yellow leaf curl virus (TYLCV) is a geminivirus with a single-stranded circular DNA genome. Its genomic DNA contains 3 short repeats, designated iterons, near the TATA box of the Replication protein (Rep) gene. Iterons of other geminiviruses are known to regulate replication through sequence-specific interactions with the cognate Reps. Here we show the role of TYLCV iterons does not require specific sequences. Rather, the secondary structure formed by two reverse-complementary iteron motifs appears to be more critical. We replaced the TYLCV genome segment encompassing all 3 iteron motifs with its counterpart in tobacco curly shoot virus, thus dramatically altering the iteron sequence. Following the fate of 18 mutants in infected plants for 9 weeks, we identified numerous de novo mutations that recovered TYLCV infections to near wild-type levels. Interestingly, these mutations did not restore the SH2 iteron sequence, thus were unlikely to reconstitute the sequence-specific iteron-Rep interaction. Rather, the mutations mostly weakened iteron-enabled DNA secondary structures. Therefore, it appears likely that strong secondary structures prevented replication of TYLCV genomes in the absence of iteron-Rep interaction. On the other hand, mutations that abolished these structures also debilitated TYLCV replication. Taken together, iterons likely regulate geminivirus replication through DNA secondary structures. Rep protein likely fine-tunes the stability of these structures through Rep-iteron interactions.