*2.5. OsAAA-ATPase1 Has ATPase Activity and Is Localized in the Cytosol*

To assess whether OsAAA-ATPase1 protein has ATPase activity, *OsAAA-ATPase1* N-terminal was fused to a His-tag and expressed in *Escherichia coli*, and purified using a high affinity Ni-resin. OsAAA-ATPase1 protein showed an ATPase activity level that was comparable to that of the positive control (potato ATPase) (Figure 6).

**Figure 6.** ATPase activity of recombinant OsAAA-ATPase1 protein. Elution buffer of Ni-resin (mock treatment), and an ATPase protein from potatoes, were used negative and positive controls, respectively.

To determine the subcellular localization of OsAAA-ATPase1 in rice cells, the EGFP-OsAAA-ATPase1 fusion protein in the rice protoplast was examined under a confocal microscope. As shown in Figure 7, EGFP-OsAAA-ATPase1 protein was co-localized with a cytosol marker, mCherry signals, indicating that OsAAA-ATPase is predominantly distributed in the cytosol.

**Figure 7.** Subcellular localization of OsAAA-ATPase1 in rice protoplasts. (**a**) EGFP-OsAAA-ATPase1, (**b**) blight field image, (**c**) mCherry (cytoplasmic localization), and (**d**) combined image of (**a**–**c**). N, nucleus; bar, 20 μm.

#### **3. Discussion**

AAA-type ATPases constitute a large protein family in a diverse range of organisms, and thus exhibit multiple and diverse cellular functions [15,33]. In plants, AAA-ATPase genes have been implicated in proteolysis [33], male meiosis [34], vacuolar maintenance [35], peroxisome biogenesis [36], morphogenesis [37], leaf senescence [29,38], and stress [28,39] and immune responses [18–22]. In this study, we present a novel rice AAA-ATPase gene member, *OsAAA-ATPase1*. The deduced amino acid sequence of OsAAA-ATPase1 contains consensus motifs that are typical of the AAA-ATPase family; these include the Walker A, Walker B, and SRH motifs (Figure 1a) [15,17,33]. Consistent with this, biochemical analysis confirmed that there was ATPase activity in the recombinant protein of OsAAA-ATPase1 (Figure 6). Phylogenetically, OsAAA-ATPase1 was grouped within a subclade of proteins related to plant defense activation (Figure 1b), which included OsAAA-ATPase2–6 [12], tobacco NtAAA1 [18,19], and *Arabidopsis* AtOM66 [20]. These results suggest that OsAAA-ATPase1 belongs to the AAA-ATPase family. Functional analysis revealed that *OsAAA-ATPase1* is transcriptionally regulated by SA in response to blast infection (Figures 2 and 3). Overexpression or RNAi-mediated suppression of *OsAAA-ATPase1* resulted, respectively, in an increase (Figure 4) or decrease (Figure 5) in blast resistance. Taken together, our results suggest that *OsAAA-ATPase1* plays a positive role in the SA-mediated disease resistance in rice plants.

In relation to plant immune responses, several studies have shown important roles for AAA-ATPase genes. *NtAAA1* was isolated as an HR-induced gene in *Nicotiana tabacum* [18]; was found to be under the control of *N*-gene, ethylene, and jasmonate; and was localized in the cytoplasm. It was also negatively involved in the SA-signaling pathway and pathogen resistance [18,19]. In contrast, *AtOM66* (outer mitochondrial membrane protein of 66 kDa ) is a stress-induced gene; overexpression of this gene increased SA content, accelerated cell death rates, and enhanced resistance to the biotrophic pathogen *Pseudomonas syringae* [20]. Recently, rice *LMR* and *LRD6-6* were map-based cloned from lesion mimic mutants *lmr* and *lrd6-6*, respectively, and were found to be the same gene (*Os06g0130000*). LMR/LRD6-6 was shown to be localized in the multivesicular bodies (MVBs) and was negatively involved in rice immunity and cell death [21,22]. Mutation in this gene (*lmr* and *lrd6-6*) resulted in constitutive expression of *PR1* and *PBZ1*, and enhanced resistance to rice blast and bacterial blight diseases; however, no difference in SA content was determined [21,22]. By comparison, it seems that OsAAA-ATPase1 plays a role distinct from those previously reported, with respect to its association with SA-regulation and HR, its subcellular localization, and its promotion of disease resistance. Thus, our findings provide novel insights into SA-regulated defense activation in rice. Meanwhile, OsAAA-ATPase1 showed a close phylogenetic association with AtOM66 (Figure 1b); both proteins play a positive role in the SA-signaling pathway, suggesting that they may share a common cellular function.

Plants produce a variety of FAs and their derivatives, some of which have been shown to play important roles in defense activation [40,41]. In the *Arabidopsis ssi2* mutant, disruption of *SSI2*, which encodes an FA desaturase, results in an increase in the 18:0 FA content, which in turn remarkably increases SA content, PR gene expression, and resistance against multiple pathogens [42]. Similar defense-related phenotypes were observed following suppression of *SSI2*-orthologs in soybean (*GmSACPD-A*/*-B*) [11], rice (*OsSSI2*) [12], and wheat (*TaSSI2*) [13,14]. These results strongly suggest that *SSI2* and its orthologs serve as valuable susceptibility gene (*S* gene) resources for the development of crop cultivars with resistance to multiple pathogens, by employing targeted mutation and genome editing technologies [43–45]. In order to make such successful use of these genes in resistance breeding, it is important to understand the molecular mechanisms underlying the defense activation. In *Arabidopsis*, a mutation in the GTPase nitric oxide associated 1 (*NOA1*) gene partially restored the *ssi2* phenotype, whereas double mutations in NOA1 and either one of the two nitrate reductase isoforms (NIA1 and NIA2) completely restored the *ssi2* phenotypes; this indicates that nitric oxide (NO) is required for constitutive defense in the *ssi2* mutant [46,47]. Nevertheless, little has been reported regarding the molecular basis of defense activation in *OsSSI2*-kd rice plants. We previously identified a group of six AAA-ATPase genes (*OsAAA-ATPase1–6*) that were upregulated in *OsSSI2*-kd rice plants [12]. In this study, all of these genes tested were induced in response to blast inoculation (Figure 3), suggesting that they each play a role in resistance to blast fungus. In contrast, *OsAAA-ATPase1–5* each exhibited a distinct induction pattern in response to different plant hormone treatments (Figure 2); *OsAAA-ATPase1* and *OsAAA-ATPase3* were induced by SA, *OsAAA-ATPase2* mainly by JA, and *OsAAA-ATPase4* and *OsAAA-ATPase5* slightly by the CK treatment. These results suggest that there is functional differentiation among the *OsAAA-ATPase1–6* genes downstream of *OsSSI2* in disease resistance. Moreover, although both *OsAAA-ATPase1* and *OsAAA-ATPase3* were induced by SA treatment, only the induction of *OsAAA-ATPase1* was attenuated following blast infection in *nahG*-rice plants (Figure 3). One possible explanation for this is that *OsAAA-ATPase3* may be more sensitive to SA, allowing it to be induced even by a residual increase in the SA-signaling level in *nahG*-rice plants.
