*2.2. OsAAA-ATPase1 is Induced by SA Treatment*

Plant hormones have been demonstrated to play important roles in interactions between plants and pathogens. Hence, we examined the transcriptional responses of *OsAAA-ATPase1–6* to the plant hormones abscisic acid (ABA), ACC (an ethylene precursor), BTH (a functional analogue of SA), kinetin (CK, a synthetic cytokinin), auxin (IAA), jasmonic acid (JA), and gibberellic acid (GA).

*OsAAA-ATPase1* (Figure 2a) and *OsAAA-ATPase3* (Figure 2c) were induced specifically by BTH treatment, and *OsAAA-ATPase2* (Figure 2b) was induced by JA treatment. Meanwhile, *OsAAA-ATPase4* (Figure 2d) and *OsAAA-ATPase5* (Figure 2e) were not specifically induced by any of the hormones, and *OsAAA-ATPase6* (*Os07g0517600*) had no detectable transcription.

### *2.3. OsAAA-ATPase1 is Induced in Response to Blast Infection in An SA-Dependent Manner*

Rice seedlings of non-transformant Nipponbare rice (NB) and of NB expressing the *nahG* gene (*nahG*-rice), at the four-leaf stage, were subjected to blast inoculation. At 2–6 days post inoculation (dpi) of the blast, the fourth leaves were sampled to examine the expression of *OsAAA-ATPase1–5*.

All of the tested *OsAAA-ATPase* genes clearly showed transcriptional induction in response to blast inoculation (Figure 3c,e,g,i); in particular, *OsAAA-ATPase1* (Figure 3a) and *OsAAA-ATPase2* (Figure 3c) showed a high-fold transcriptional increase from the very low basal levels in the mock treatment. The induction of the genes became evident from 2 dpi and peaked at ca. 3–5 dpi.

In *nahG*-rice plants, in contrast, the induction of *OsAAA-ATPase1* was mostly attenuated relative to its induction in NB plants in response to blast inoculation (Figure 3b), demonstrating that the induction of this gene depends on the SA-signaling pathway. No appreciable attenuation of gene induction was observed for the other genes (Figure 3d,f,h,j).

#### *2.4. OsAAA-ATPase1 is Positively Involved in Blast Resistance*

To gain some insight into the role of *OsAAA-ATPase1* in disease resistance, we generated transgenic rice lines that either overexpressed the gene under maize ubiquitin promoter (*OsAAA-ATPase1*-ox; Figure 4a) or RNAi-suppressed *OsAAA-ATPase1* (*OsAAA-ATPase1*-kd; Figure 5a), and subjected these lines to blast inoculation. In order to reveal the potentially compromised resistance in *OsAAA-ATPase1*-kd plants, a half density of conidia (5 <sup>×</sup> 104/mL) was used, so as to cause blast disease moderately in NB, but more severely in OsAAA-ATPase1 plants.

**Figure 4.** Blast resistance of *OsAAA-ATPase1*-ox plants. (**a**) Expression of *OsAAA-ATPase1*, (**b**) blast lesions on leaf blades, and (**c**) relative fungal growth (*Magnaporthe oryzae rDNA*). (**d**,**e**) Expression of *PR1b* (**d**) and *PBZ1* (**e**), in Nipponbare (NB) and *OsAAA-ATPase1*-ox lines (#27 and #29), respectively, at 7 days post inoculation (dpi). Data are represented as means ± SDs in (**a**,**c**–**e**).

**Figure 5.** Compromised blast resistance in *OsAAA-ATPase1*-kd plants (#25 and #32). (**a**) Expression of *OsAAA-ATPase1*. (**b**) Relative fungal growth (*M. oryzae rDNA*) in Nipponbare (NB) and *OsAAA-ATPase1*-kd lines (#25 and #32) respectively. Data are represented as means ± SDs.

Compared with the non-transgenic control plants (NB), *OsAAA-ATPase1*-ox plants (lines #27 and #29) exhibited significantly higher resistance to blast disease, as evidenced by the fact that few susceptible blast lesions appeared on their leaf blades (Figure 4b), and that they had ca. 4-fold less fungal growth (Figure 4c). The enhanced resistance of the *OsAAA-ATPase1*-ox plants is consistent with the large increases in the expression levels of the PR genes, *OsPR1* and *PBZ1* (Figure 4d,e).

Conversely, blast resistance was significantly compromised in *OsAAA-ATPase1*-kd plants (lines #25 and #32): they had ca. 2-fold more fungal growth than the NB control plants (Figure 5b).
