**1. Introduction**

Salicylic acid (SA) plays an important signaling role in plant defense activation against pathogens. In response to pathogen attack, SA activates a battery of defense-related genes, including pathogenesis-related (PR) genes, throughout the plant, resulting in both local and systemic resistance to the pathogen [1]. In *Arabidopsis*, NPR1 (non-pathogenesis related 1) has been demonstrated to play a master role in SA-mediated defense activation [2,3]. A loss of NPR1 function (npr1) results in loss of PR gene induction, and hypersensitivity to diseases [4]. In rice, meanwhile, it has been shown that SA signaling is mediated by two downstream factors, OsNPR1 and WRKY45, acting in parallel [5,6].

In *Arabidopsis*, genetic screening for mutations that can suppress *npr1* phenotypes (based on their ability to restore SA-induced PR expression to npr1-5 plants) resulted in isolation of several *npr1* suppressor mutants (*ssi*: suppressor of SA insensitivity), which exhibit constitutive defense activation [7]. Map-based cloning of one of the *ssi* mutants (*ssi2*) revealed that the corresponding gene (*SSI2*) encodes a stearoyl-ACP desaturase, which desaturates stearoyl (18:0)-ACP into oleoyl-ACP, and finally, into oleic acid (18:1) [7]. Disruption of this gene in *ssi2* results in a ten-fold increase in 18:0 fatty acid (FA) content, indicating involvement of FAs in plant defense reactions [7]. The *ssi2* mutant plants accumulate high levels of SA and display constitutive PR gene expression and enhanced resistance to *Peronospora parasitica*, *Pseudomonas syringae* [8], and *Cucumber mosaic virus* [7,9,10].

The orthologs of *SSI2* have also been identified in soybean (*GmSACPD-A*/*-B*) [11], rice (*OsSSI2*) [12], and wheat (*TaSSI2*) [13,14]. Similar to *Arabidopsis ssi2*, suppression of these ortholog genes enhanced resistance to multiple pathogens: *Pseudomonas syringae* pv. *glycinea* and *Phytophthora sojae*in soybean [11]; blast fungus *Magnaporthe oryzae* and leaf-blight bacteria *Xanthomonas oryzae* pv. *oryzae* (*Xoo*) in rice [12]; and powdery mildew bacteria *Blumeria graminis* f. sp. *tritici* and Fusarium head blight fungus *Fusarium graminearum* in wheat [13,14]. These results demonstrate a common function of *SSI2* and its orthologs in defense activation in diverse plant species.

The molecular mechanisms whereby the *SSI2* family genes participate in defense reactions in plants remain to be fully elucidated. In rice, a DNA microarray analysis revealed several hundred genes differentially expressed between the wild-type and *OsSSI2*-suppressed transgenic (*OsSSI2*-kd) plants [12]. Among them was a group of six genes for AAA-ATPase (AAA: ATPases associated with diverse cellular activities) highly upregulated in *OsSSI2*-kd plants, in addition to the well-known defense-related genes, such as *WRKY45*, *PR1b*, and *PBZ1*, and a *thaumatin-like* gene [12]. These results suggest that the AAA-ATPase family genes may play important roles in defense activation in rice plants.

The AAA-ATPase family occurs in all life forms, including eukaryotes, prokaryotes, and archaebacteria, and is implicated in a variety of cellular activities, including proteolysis, protein folding, membrane trafficking, cytoskeletal regulation, organelle biogenesis, DNA replication, and immune responses [15–17]. Structurally, these proteins contain one or several conserved motifs, including the Walker A and Walker B motifs, which are, respectively, required for ATP binding and hydrolysis; they also contain a highly conserved amino acid sequence, referred to as the second region of homology (SRH) [16]. In plants, it has been reported that AAA-ATPase genes from *Nicotiana tabacum* (*NtAAA1*) [18,19] and *Arabidopsis* (*AtOM66*) [20] are, respectively, negatively or positively involved in the SA-signaling pathway and in the hypersensitive response (HR) upon pathogen infections. Moreover, in rice, map-based cloning of the *lesion mimic resembling* (*lmr*) mutant/*lesion resembling disease* (*lrd6-6*) mutant revealed that the corresponding gene (*LMR*/*LRD6-6, Os06g0130000*) encodes an AAA-ATPase, and is negatively involved in HR and disease resistance [21,22].

In this study, we conducted a functional analysis of *OsAAA-ATPase1*, one of the six AAA-ATPase genes upregulated in *OsSSI2*-kd rice plants [12]. We show that *OsAAA-ATPase1* is transcriptionally regulated by SA, and positively involved in resistance to blast fugus *M. oryzae*.

#### **2. Results**

In our previous study, a group of six AAA-ATPase family genes (hereafter *OsAAA-ATPase1–6*; (Table 1; Figure 1) was found to be significantly upregulated in *OsSSI2*-kd rice plants [12], implicating these genes in rice defense activation. From among them, we chose *OsAAA-ATPase1* for more detailed functional characterization in this study, because it showed SA-induced (Figure 2) and SA-dependent blast-induced (Figure 3) transcription responses.


**Table 1.** Genes and primer sequences used for qRT-PCR analysis.
