*2.1. OsAAA-ATPase1 Encodes an AAA-ATPase Family Protein*

*OsAAA-ATPase1* was predicted to encode a protein of 520 amino acids with a predicted mass of 58.1 kDa. OsAAA-ATPase1 shared 50%, 37.8%, and 22.3% amino acid sequence identity with previously reported defense-related AAA-ATPase proteins, namely, tobacco NtAAA1 [18], *Arabidopsis* AtOM66 [20], and rice LMR/LRD6-6 [21,22], respectively. Structural analysis revealed that OsAAA-ATPase1 contains consensus motifs that are typical of the AAA-ATPase family; these include the Walker A, Walker B, and SRH motifs (pfam00004; E-value <sup>=</sup> 4.95 <sup>×</sup> <sup>10</sup><sup>−</sup>17) (Figure 1a).

To assess how *OsAAA-ATPase1*–*6* are related within the AAA-ATPase gene family, we performed a phylogenic comparison of the proteins predicted to be encoded by *OsAAA-ATPase1–6* and several known rice AAA-ATPase proteins, including LMR/LRD6-6 [21,22], OsCDC48 [27], RuvBL1a [28], RLS3 [29], OsSKD1 [30], OsFtsH5 [31], and RFC5 [32], together with NtAAA1 [18,19] and AtOM66 [20]. OsAAA-ATPase1 was grouped within a subclade of proteins related to plant defense, including OsAAA-ATPase2–6, NtAAA1, and AtOM66, but, unexpectedly, distally with LMR/LRD6-6 (Figure 1b).

**Figure 1.** Amino acid sequence alignment of AAA-type ATPase proteins, and phylogenetic analysis. (**a**) Alignment of typical consensus motifs of the AAA-ATPase protein family, including the Walker A, Walker B, and SRH motifs. (**b**) Phylogeny of the AAA-ATPase proteins from rice (LMR/LRD6-6, OsCDC48, RuvBL1a, RLS3, OsSKD1, OsFtsH5, and RFC5), tobacco (NtAAA1), and *Arabidopsis* (AtOM66).

**Figure 2.** Expression analysis of *OsAAA-ATPase1–5* (**a**–**e**) in response to the plant hormones abscisic acid (ABA), ethylene (ACC, an ethylene precursor), benzothiadiazole (BTH, a functional analogue of SA), kinetin (CK, a synthetic cytokinin), auxin (IAA), jasmonic acid (JA), and gibberellic acid (GA), in Nipponbare rice seedlings. Data are represented as means ± SDs.

**Figure 3.** Expression analysis of *OsAAA-ATPase1–5* (**a**–**j**) in response to blast inoculation in Nipponbare (NB) and *narG* rice seedlings. Rice seedlings at the four-leaf stage (three true leaves) were subjected to mock treatment (mock) or blast inoculation (blast-inoculated), and the fourth leaf blades were sampled at indicated days post inoculation (dpi). Data are represented as means ± SDs.
