Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1–6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice’s defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.


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].

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.

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 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).

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 × 10 4 /mL) was used, so as to cause blast disease moderately in NB, but more severely in OsAAA-ATPase1 plants.   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).

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). 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.
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][44][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.

Plant Materials and Growth Conditions
Japonica type rice cultivar Nipponbare (Oryza sativa L.) was grown in commercial nursery soil (Bonsol Number 2; Sumitomo Chemical Corp., Tokyo, Japan) in a greenhouse at 28 • C (day)/23 • C (night) with ca. 50% relative humidity.

Plasmid DNA Construction and Rice Transformation
The cDNA clone for OsAAA-ATPase1 was provided by the Rice Genome Resource Center, Japan (accession number: AK070731). To construct a plasmid for constitutive expression of OsAAA-ATPase1 under the maize ubiquitin promoter, a DNA fragment containing a 91 bp upstream sequence followed by the full coding sequence of OsAAA-ATPase1 (nucleotides 2-1655) was amplified by PCR and cloned into the pUCAP/Ubi-NT vector, as previously described [5]. To construct a plasmid for OsAAA-ATPase1 RNAi (OsAAA-ATPase1-kd), part of the 3 -UTR (nucleotides 1543-1845) of OsAAA-ATPase1 cDNA was amplified by PCR and cloned into the pANDA vector, as previously described [48,49].
Nipponbare rice plants were transformed by an Agrobacterium tumefaciens (strain EHA105) mediated technique, as described earlier [50]. Transgenic rice lines expressing nahG from Pseudomonas putida under the control of a double 35S promoter (nahG-rice) were generated using the plant expression construct previously described in Yang et al. [51].
For plant treatments, rice seedlings at the four-leaf stage (three true leaves) were transferred to a container containing each of the plant hormone solutions at 50 µM. The rice seedlings were further grown for 1 day, and fourth leaf blades were stored in liquid nitrogen for RNA preparation.

Protein Expression, Purification, and ATPase Assay
The OsAAA-ATPase1 sequence was amplified by PCR and cloned into the sites between BglI and HidIIIp of pET32a (Novagen) as a His-tag fusion protein; then, that was transfected into the Escherichia coli Origami strain BL21(Lys).
The set of primers used was as follows: OsAAA1BglII 5 -GTAGATCTCTTGAGACAAATGGAGGCGACG-3 ; OsAAA1HindIII 5 -GCTAAGCTTCTACTTATCCTTCCCGACCAC-3 . Expression of the protein was induced for 4 h at 25 • C with 0.5 mmol/L isopropyl β-d-1-thiogalactopyranoside. Escherichia coli cells were pelleted by centrifugation, resuspended in lysis buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 10 mmol/L imidazole), and sonicated. After the cell debris was removed by centrifugation (12,000 × g, 10 min, 4 • C), the supernatant was loaded onto a High Affinity Ni-Charged Resin (GE Healthcare, Buckinghamshire, UK), washed with washing buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 10 mmol/L imidazole), and eluted with elution buffer (20 mmol/L Tris-HCl pH 7.4, 0.1 M NaCl, 180 mmol/L imidazole). ATPase activity was measured by the malachite green-based colorimetric method using the QuantiChrom TM ATPase/GTPase activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). The elution buffer was used as the negative control, and an ATPase from potatoes (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control. One unit is defined as the amount of enzyme that catalyzes the production of 1 µM of free phosphate per minute under the assay conditions.

Subcellular Localization
For subcellular localization of OsAAA-ATPase1, the plasmid pSAT6-AFP-C1-OsAAA1 was transformed into protoplasts prepared from etiolated seedlings as previously described [52]. As a control for cytoplasmic localization, the pSAT-mCherry construct was co-transformed. Fluorescence was examined under a confocal microscope (Leica Microsystems, Wetzlar, Germany) 16 h after transformation.

Pathogen Culture and Inoculations
Culture and inoculation of the blast fungus M. oryzae (compatible race 007.0) was conducted essentially as previously described [12], with slight modifications. Briefly, the fungus was grown on an oatmeal agar medium (30 g/L oatmeal, 5 g/L sucrose, and 16 g/L agar) at 26 • C for 10-12 day. After removing the aerial hyphae by washing with distilled water and a brush, conidia formation was induced by irradiation under continuous black blue light (FL15BLB; Toshiba, Osaka, Japan) at 24 • C for 3 day. The conidia were suspended in 0.02% Silwet L-77 (a non-ionic surfactant; Nihon Unica, Tokyo, Japan) at a density of 10 5 /mL, and were sprayed onto rice plants at the four-leaf stage. After incubation in a dew chamber at 24 • C for 24 h, the rice plants were moved back to the greenhouse.

RNA Analyses
Total RNA was isolated from leaf blades of the 4th leaves of rice seedlings using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR (qRT-PCR) was performed on a Thermal Cycler Dice TP800 system (Takara Bio, Tokyo, Japan) using SYBR premix Ex Taq mixture (Takara Bio) as previously described [5]. The primer sequences used for qRT-PCR are listed in Table 1.

Amino Acid Sequence Alignment and Phylogenetic Analysis
The protein sequences were retrieved from the rice annotation project database (rap-db) and aligned using Clustal-X software, and the tree was constructed using iTOL software [53].

Conclusions
In this study, we present a novel AAA-ATPase member gene, OsAAA-ATPase1, one of the six AAA-ATPase genes upregulated in OsSSI2-kd rice plants [12]. Functional analysis revealed that OsAAA-ATPase1 is transcriptionally regulated by SA, and plays a positive role in the SA-mediated disease resistance in rice plants. Our findings provide novel insights into SA-regulated defense activation in rice, and the molecular basis of defense activation in OsSSI2-kd rice plants.