Disequilibration of Pik-H4 expression did not affect rice blast resistance
The Pik gene pairs trigger HR together facing Avr and form a complex (ZdrzaƂek et al., 2020a). Considering the co-regulation of thePik-H4 gene pair via the BDP, we investigated whether the disequilibration of Pik-H4 expression had any influence on rice blast resistance. Remarkably, the overexpression of eitherPik1-H4 or Pik2-H4 in LTH did not confer disease resistance (Figure 5A-C). Conversely, withinPik-H4 NILs, overexpression of eitherPik1-H4 or Pik2-H4sustained rice blast resistance. Interestingly, overexpression of eitherPik-H4 in LTH led to an enhancement in the expression of the other Pik gene in LTH (Figure 5D). The relative expression levels analysis showed overexpressing one of the Pik-H4 in Pik-H4NILs up-regulated the counterpart (Figure 5E). This intriguing regulatory relationship was successfully replicated in tobacco, yielding analogous outcomes (Figure 5F). These findings collectively suggest that, facilitated by the PPik-H4 , the expression levels of Pik-H4 are self- and co-regulated. To eliminate the innate regulation of Pik-H4 in rice, agroinfiltrations of different ratios of Pik-H4 with 35S promoter were performed in tobacco (Figure 5G). At the protein level, combinations of Pik1-H4 and Pik2-H4 in 1:1, 1:2, or 2:1 ratios failed to induce HR in the absence of AvrPik-E. However, upon the introduction of AvrPik-E, these combinations exhibited activity in ion leakage assays, with similar conductivity levels recorded at 3 dpi. These results suggested that altering the disequilibration ofPik-H4 in protein levels did not compromise rice blast resistance.