The characterization of Pik-H4 promoter
The Pik genes, such as Pik-H4 (Figure 1A), consist of two adjacent and H2H NLRs sharing a promoter region. The promoter region ofPik-H4 (PPik-H4 ) was cloned and aligned to that of Pik -Nip and Pikh (Figure S1). The sequence similarity of PPik-H4 (2492 bp) and PPikh (2542 bp) is 97.44%, and the major sequence differences are located between 1010 bp to 1400 bp of PPik-H4 , which results in lacking 8 TFBSs compared to PPikh . PPik-H4and PPik-Nip (1099 bp) only share 25.41% similarity, and the promoter sequences of the Pik-1 direction are highly dissimilar. Interestingly, there are two types of promoter length of available promoter sequences of reported Pik genes.Pik* , Pi1 , Pik-h , andPik-m promoter regions are 2542 bp in length, while those ofPi7 , Pik-H4 , and Pikp are approximately 2492 bp. All these promoters are highly conserved even though these Pikgenes were cloned from different donors (Table S1). Then, the functional motifs of PPik-H4 were detected (Figure 1B). Ten putative TATA boxes were predicted in PPik-H4 , and 3 of them were bidirectional TATA boxes. The bidirectional TATA box (5’-TATATAT-3’) confers bidirectional transcriptional activity and is highly active (Xu et al., 1991). Therefore, the bidirectional TATA box may enable the PPik-H4 transcript flanking genes. Besides, TFBSs of well-known immunity-related TFs (ERF, WRKY and Dof) were also found in PPik-H4 , indicating the pathogen-induced possibility of Pik-H4 .