Functional validation of the Pi54 gene by knocking down its expression in a blast-resistant rice line using RNA interference and its effects on other traits.

Rice blast disease caused by Magnaporthe oryzae is one of the major diseases affecting the rice (Oryza sativa L.) crop. A major blast resistance gene, Pi54, has already been cloned and deployed in different rice varieties. To understand the role of Pi54 in providing rice blast resistance, we used the RNA interferences (RNAi) approach to knock down the expression of this gene. We showed a high frequency of Agrobacterium tumefaciens-mediated transformation of rice line Taipei 309 containing a single gene (Pi54) for blast resistance. Pi54 RNAi leads to a decreased level of Pi54 transcripts, leading to the susceptibility of otherwise M. oryzae-resistant rice lines. However, among the RNAi knockdown plants, the severity of blast disease varied between the lines. Histochemical analysis of the leaves of knockdown plants inoculated with M. oryzae spores also showed typical cell death and blast lesions. Additionally, Pi54 RNAi also showed an effect on the Hda3 gene, a florigen gene playing a role in rice flowering. By using the RNAi technique, for the first time, we showed that the directed degradation of Pi54 transcripts results in a significant reduction in the rice blast resistance response, suggesting that RNAi is a powerful tool for functional validation of genes.