Functional characterization of OsRacB GTPase – a potentially negative regulator of basal disease resistance in rice

Plant Physiology and Biochemistry 44 (2006) 68–77 www.elsevier.com/locate/plaphy

Original article

Functional characterization of OsRacB GTPase – a potentially negative regulator of basal disease resistance in rice Young-Ho Jung a, Ganesh Kumar Agrawal b, Randeep Rakwal b,c, Jung-A Kim a, Mi-Ok Lee a, Pil Gyu Choi a, Young Jin Kim d, Min-Jea Kim e, Junko Shibato c, Sun-Hyung Kim f, Hitoshi Iwahashi c, Nam-Soo Jwa a,* a Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), GPO Box 8207, Kathmandu, Nepal c Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan d School of Life Sciences and Biotechnology, Korea University, Anam-dong, Sungbuk-gu, Seoul 136-701, Korea e Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women’s University, Seoul 139-774, Korea f Research Institute of Agricultural Resources, Ishikawa Agricultural College, Suematu, Nonoichi, Ishikawa 921-8836, Japan b

Received 17 March 2005; received in revised form 18 August 2005; accepted 22 December 2005 Available online 17 February 2006

Abstract The rice genome contains at least seven expressed Rop small GTPase genes. Of these Rops, OsRac1 is the only characterized gene that has been implicated in disease resistance as a positive regulator. To our interest in finding a negative ROP regulator of disease resistance in rice, we applied a “phylogeny of function” approach to rice Rops, and identified OsRacB based on its close genetic orthologous relationship with the barley HvRacB gene, a known negative regulator of disease resistance. To determine the function of OsRacB, we isolated the OsRacB cDNA and conducted gene expression and transgenic studies. OsRacB, a single copy gene in the genome of rice, shared 98% identity with HvRacB at the amino acid level. Its mRNA was strongly expressed in leaf sheath (LS) and in panicles, but was very weakly expressed in young and mature leaves. The basal mRNA level of OsRacB in LS of two-week-old seedlings was strongly down-regulated upon wounding by cut and treatment with jasmonic acid. A dramatic down-regulation in the OsRacB transcripts was also found in plants inoculated with the blast pathogen, Magnaporthe grisea. Interestingly, transgenic rice plants over-expressing OsRacB showed increased symptom development in response to rice blast pathogens. Additionally, fluorescence microscopy of green fluorescent protein (GFP):OsRacB-transformed onion cells and Arabidopsis protoplasts revealed OsRacB association with plasma membrane (PM), suggesting that PM localization is required for proper function of OsRacB. Based on these results, we suggest that OsRacB functions as a potential regulator for a basal disease resistance pathway in rice. © 2006 Elsevier SAS. All rights reserved. Keywords: Barley; Disease resistance; Magnaporthe grisea; Oryza sativa; ROPs; Signaling

1. Introduction The ROP proteins of plants are small GTPases, and are designated `Rop’ (Rho-related GTPase from plant), as they share Abbreviations: CaMV, cauliflower mosaic virus; GFP, green fluorescent protein; HR, hypersensitive resistance; JA, jasmonic acid; MS, Murashige and Skoog; ORF, open reading frame; PCR, polymerase chain reaction; ROS, reactive oxygen species. * Corresponding author. E-mail address: [email protected] (N.-S. Jwa). 0981-9428/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.12.001

a slightly higher overall similarity with mammalian Rac, a subfamily of Rho GTPase [22–25,27]. Like the mammalian Rho GTPase family, the members of the plant Rop family are activated by upstream signals, and are thought to involve at least three regulators, the guanine nucleotide exchange factors (GEFs), the GTPase-activating proteins (GAPs), and the guanine nucleotide dissociation inhibitors (GDIs) [10,22,23,27]. ROPs cycle between two conformational states: ROP bound to GTP (guanosine triphosphate) is an active state, whereas ROP bound to GDP (guanosine diphosphate) is an inactive state. Activation of the GTPase, through GDP-GTP exchange,

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is promoted by GEFs, whereas inactivation (by an intrinsic GTPase activity) is stimulated by GAPs. ROP-GDI appears to stabilize the inactive GDP-bound form of the protein. Activated ROP GTPases interact with one or more downstream cellular target proteins and produces a variety of cellular responses. ROPs have emerged as an important molecular switch in plant signal transduction [1,9,27]. They have been found to regulate a diverse array of cellular responses, such as cell polarity/tip growth, actin cytoskeleton, secondary wall formation, and meristem signaling [9,27]. Further interest in ROPs is fuelled by recent studies, which unveil a decisive role of ROPs in the plant disease resistance mechanisms [1]. Importantly, most of these disease resistance studies have mainly been conducted in monocotyledonous (monocot) plants, such as rice (Oryza sativa L.) and barley (Hordeum vulgare L.). It is important to mention that in several literatures, the Rop gene has been termed as the Rac gene, and therefore, for clarity we have used the same gene name throughout this manuscript. In rice, over-expression of the constitutive active form of OsRac1 leads to hypersensitive resistance (HR) at the sites of attack by rice blast fungus (Magnaporthe grisea, race 007) and, therefore, to pathogen resistance via reactive oxygen species (ROS) production. On the other hand, over-expression of dominant negative forms of OsRac1 consistently results in enhanced susceptibility to M. grisea [12,16]. A similar result is also obtained when these transgenic plants were challenged by a bacterial pathogen, Xanthomonas oryzae, which causes blight disease [16]. Furthermore, OsRac1 has been found to suppress the expression of genes encoding metallothioneins that scavenge ROS [26]. At the protein and activity levels, a study by M. Luo in 2003 reported the prokaryotic expression and characterization of rice rac protein OsRACB by inserting the complete coding sequence of OsRACB into a pET28a expression vector followed by expression in E. coli BL21 cells [15]. Using the purified and active OsRACB protein, and performing the GTPbinding activity and hydrolyzing assay, it was found that OsRACB has strong GTP special binding and hydrolysis activity regulated by Mg2+. They further showed that OsRACB has stronger GTP-binding activity and a weaker hydrolysis activity when compared to OsRACD. This study clearly demonstrates the functional aspect of OsRACB in rice. In all, the above findings [12,16,26] suggest that OsRac1 acts as a positive regulator of a disease resistance pathway functioning downstream of R genes. In barley, two studies have brought an interesting twist in regulation of the disease resistance pathways by ROPs, where ROPs are implicated in susceptibility of plant to invasion by a powdery mildew pathogen Blumeria graminis f. sp. hordei (Bgh) [20,21]. It has been shown that HvRacB is required for successful establishment of fungal haustorium [20]. In a detailed functional study (transient knock-down and over-expression), it has been observed that first, barley leaves transformed with HvRacB-hairpin construct [Pro35S:HvRacB(antisense)-Intron-HvRacB(sense)] have reduced penetration efficiency of Bgh, and second, over-expression of constitutive active form of OsRacB (HvRacB-V15) con-

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sistently results in increased susceptibility to invasion of barley leaves by Bgh [20,21]. The same group has also identified at least two other barley ROPs, HvRac3 and HvROP6, and has shown their potential role in the establishment of susceptibility to Bgh [21]. These initial but important studies, conducted by two independent groups, lead us to assume that plant disease resistance signaling pathways possess positive and negative ROP components that might employ both components for its fine-tuned regulation against pathogens. If this is the case, rice and barley should also possess a negative and positive ROP regulator, respectively, for a disease resistance pathway. To test our above assumption, we selected rice as a model system, and applied a “phylogeny of function” approach in combination with gene expression and transgenic studies. As a proof-of-concept, we identify a Rop gene from rice, called OsRacB, and provide evidence that OsRacB acts as a negative regulator of the basal disease resistance pathway. 2. Results and discussion 2.1. Phylogeny of function approach identifies OsRacB as a possible negative regulator of disease resistance In barley, ROPs (HvRacB, HvRac3 and HvROP6) were found to be involved in increased disease susceptibility to the powdery mildew fungus [20,21]. This finding suggests the existence of negative ROP regulators for the disease resistance pathways in plants. As we have been using rice as a model cereal crop for understanding plant cell signaling during development and defense response, and as OsRac1 is the only rice Rop gene so far functionally implicated as a positive regulator of disease resistance pathway(s), we embarked to search for a negative ROP regulator in rice. We used a phylogeny of function approach to find rice ROP(s) that form a group (or subgroup) with HvRacB, HvRac3, or HvROP6. To do so, we constructed a phylogenetic tree with ROPs derived from rice, barley, maize, and Arabidopsis (data not shown, see reference [1]). It was found that OsRacB, OsRac3, and OsRac2 form a group with HvRacB, HvRac3, and HvRop6, respectively. Of these, OsRacB appears to be unique, as its group is composed of only monocotyledonous rops, HvRacB and ZmRop2 [1]. This result is in line with previous finding that OsRacB belongs to group 4 and forms a unique subgroup with only monocot ROPs in extended and refined phylogenetic analyses of Rop genes [8]. Based on the tenet of phylogeny of function – genes of similar function are likely to group together when arranged in a phylogenetic tree [17] – we assumed that OsRacB might have a function similar to that of HvRacB in disease resistance. In order to illustrate the function of OsRacB, we cloned the OsRacB cDNA using a cDNA library prepared from total RNA of rice panicle before heading (PBH). We used the cDNA library of rice PBH, because several Arabidopsis ROPs are highly expressed in pollens [14]. The OsRacB cDNA was 1066 bp long, which possessed the longest ORF of 590 bp,

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encoding a 197 amino acid polypeptide with a predicted molecular mass of 21623.61 and a pI of 9.4 (Compute pI/MW tool, ExPASy). OsRacB showed 98% sequence identity to HvRacB at the amino acid level (data not shown), indicating that they are orthologous and structurally related genes. OsRacB also possessed all the structural features of plant ROPs, such as ATP/GTP-binding site motif (P-loop, amino acids 13-20, GDGAVGKTCML), tyrosine kinase phosphorylation site (TKPS, amino acids 99-107), prenylation site (CAAX box; amino acids 753-983), and C-terminal hypervariable region (HVR; PKAKKKKKAQRGACSIL). Like ZmRop2, OsRacB carried an extraordinarily long 3′-untranslated region (3′UTR) of ca. 381 bp and shared high sequence identity (83%) at the nucleotide level (Fig. 1A and B). In contrast, the 3`UTRs of OsRacB shared very low sequence identity (5-20%) with other rice ROPs. Furthermore, genomic Southern analysis conducted on total genomic DNA digested with restriction enzymes (HindIII, EcoRI, XbaI, BglII, BamHI and PstI) indicated that OsRacB is a single copy gene in the rice genome (data not shown). 2.2. OsRacB is developmentally regulated To investigate spatial and temporal distribution of OsRacB mRNA during the course of rice growth and development, total RNA from young leaf (L) and leaf sheath (LS) of fourteen-day-

old seedlings, and the flag (FgL) and first leaf (FL), and panicles of different stages, such as PBH, panicle after heading (PAH), and at maturity (PAM), of mature plants was subjected to northern blot analysis using the OsRacB specific gene probe (Fig. 2A). The results showed that OsRacB gene was expressed in all rice tissues or organs investigated. OsRacB mRNA was weakly expressed in young seedling leaves (L) and matured FL and FgL. The OsRacB mRNA, on the other hand, was strongly expressed in LS, and PBH, PAH, & PAM (pollination stage). These results demonstrated that OsRacB transcripts are constitutively and differentially expressed in various tissues and organs at different developmental stages of rice, suggesting a constitutive and/or specific function of OsRacB throughout the whole plant lifetime. Additionally, the preferential expression of OsRacB in the more actively dividing and/or expanding tissues or organs (such as young LS and developing panicles) implied that OsRacB may play an important role in fast-growing tissues and organs, and may be differently regulated in response to environmental cues. Our expression data are in general agreement with a more detailed expression study on nine Rop genes in maize, where mRNA levels of most of the Rops were found to be significantly lower, compared with shoot apex [8]. Developmental regulation of the barley HvRacB is still unknown, and therefore, it would be interesting to see whether HvRacB possesses expression patterns similar to OsRacB.

Fig. 1. Nucleotide sequence alignment of rice OsRacB 3`-UTR with the closely related HvRacB and ZmRop2 3`-UTRs. Alignment (A) and homology (B) of nucleotide sequence was done using the MultAlin 5.4.1 (INRA) and CLUSTAL W (1.81) programs at ExPASy (www server). Nucleotide accession numbers are given in parenthesis besides the names of the genes in B.

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Fig. 2. Developmental and transient regulation of the OsRacB mRNA expression in rice tissues. A, Total RNA was extracted from leaf segments. Young leaf (L) and leaf sheath (LS) of fourteen-day-old seedlings; flag (FgL) and first leaf (FL), panicle before heading (PBH), after heading (PAH), and at maturity of pollen (PAM) of mature plants. B, Leaf segments treated with wounding by cut (CUT) and 100 μM JA. Sampling times are indicated above each lane, respectively. The blots were hybridized to a [α-32P]dCTP-labeled OsRacB cDNA probe. Equal loading (20 μg) was confirmed by staining of membranes with ethylene bromide (EtBr); a part of rRNA is shown. Northern analysis was carried out as described in Materials and methods.

2.3. Down-regulation of OsRacB by signaling molecules, chemical elicitors, and blast pathogens To gain insight into how the OsRacB expression is modulated by diverse signaling molecules or chemicals, we used our established two-week-old rice seedling in vitro model system [2–4]. As OsRacB is strongly expressed in young LS, we determined the OsRacB expression in LS after wounding by cut (CUT) and upon jasmonic acid (JA) treatment, as a first step (Fig. 2B). As expected, wounding by cut, the most common stress, dramatically down-regulated the OsRacB expression within 3 h, which was followed by a gradual decrease in its expression level till 24 h. In case of JA treatment, on the other hand, a more severe effect on down-regulation of OsRacB was seen, where the effect was more profound even at 3 h after JA treatment, compared with wounding by cut. As wounding by cut and JA are known to regulate defense signaling in rice [2– 4], and in plants [5,6,19], these results indicated that OsRacB is down-regulated during defense signaling. To further strengthen these results, we treated young leaves with a variety of signaling molecules [like JA, salicylic acid (SA), abscisic acid (ABA), and ethylene-generator ethephon (ET)] and chemical elicitors such as the protein phosphatase inhibitors, cantharidin (CN) and endothall (EN), for a period of 30 min to 2 h, and 3 to 72 h. These treatments have previously been shown to elicit defense/stress response in rice [2–4,18]. Almost no increase in mRNA abundance with any of these treatments was observed (data not shown).

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Now to know whether or not the M. grisea blast pathogens down-regulate the OsRacB transcripts, wild-type Nipponbare seedlings were inoculated with compatible (race 007) and incompatible (race S102) blast pathogens, as mentioned in the Materials and methods section. In this particular set of experiments, we employed a sensitive method, called reverse transcriptase polymerase chain reaction (RT-PCR), to examine changes in mRNA expression levels in leaves mainly due to low level expression of OsRacB in leaf, as detected by northern blot analysis. As we expected, the OsRacB mRNA was dramatically down-regulated in leaves infected with compatible and incompatible pathogens compared to the healthy leaves at the start of the experiment (Fig. 3). In parallel we also examined the response of a rice pathogenesis-related marker gene, OsPR1b [3]. OsPR1b was induced more rapidly and its mRNA accumulated at significantly higher levels by incompatible pathogen than that of compatible pathogen. In all, down-regulation of the OsRacB expression by wounding, signaling molecules, chemical elicitors, and more importantly, also by both compatible and incompatible blast pathogens suggests that OsRacB is perhaps a negative regulator of basal disease resistance pathway(s). 2.4. OsRacB over-expression promotes susceptibility to rice blast pathogens To get functional evidence on OsRacB association with disease resistance pathway(s), we applied an over-expression transgenic approach in rice (cv. Nipponbare). Transgenic plants generated with vector and Pro35S:OsRacB constructs were called vector and Pro35S:OsRacB plants, respectively (Fig. 4A). Vector plants served as an appropriate control for Pro35S:OsRacB plants, over-expressing the OsRacB gene under the control of CaMV 35S promoter. In general, Pro35S:OsRacB plants were phenotypically very close to vector plants. However, some Pro35S:OsRacB plants showed slightly reduced height and early flowering. As shown in Fig. 4B, we selected five independent lines (R1 to R4, and R6) of Pro35S:OsRacB plants, based on OsRacB expression level in young leaves (Fig. 4B). Of these lines, R4 had the lowest level of OsRacB expression. The OsRacB expression was almost undetectable in the leaves of vector plants. To assess the response of these transgenic lines to rice blast pathogens, twenty transgenic plants from each line were subjected to compatible (007 and KI1117) and incompatible (S102) infections. In repeated experiments, Pro35S:OsRacB plants were found to be more susceptible to compatible blast infection than vector plants (Fig. 5A). Disease appearance in vector plants was delayed by 12 to 24 h compared to Pro35S:OsRacB plants. Disease severity evaluated for 007 and KI1117 pathogens at 72 h (race 007, and S102; 4 days post-inoculation, KI1117; 6 days postinoculation) post-inoculation showed an increased disease progression in Pro35S:OsRacB plants by at least 2-fold, depending on individual lines (Fig. 5B and C). On the other hand, the HRlike lesions caused by incompatible blast pathogen (S102) in Pro35S:OsRacB plants were found to be relatively bigger in size

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Fig. 3. The OsRacB mRNA expression is strongly down-regulated by compatible and incompatible rice blast pathogens M. grisea. Nipponbare seedlings were inoculated with compatible (007) and incompatible (S102) blast pathogens, as described in section 4.8. Total RNA extracted from leaves before (0 days) and after (2, 3, and 4 days) pathogen inoculation was subjected to RT-PCR to monitor changes in mRNA expression levels of OsRacB and OsPR1b. Two independent experiments were carried out, and a representative gel image is presented. Histograms below each gel image represent relative intensity of mRNA level.

that OsRacB is a negative component of a basal disease resistance pathway for the blast pathogens. 2.5. OsRacB localizes to the plasma membrane

Fig. 4. Analysis of transgenic rice plants expressing the constitutively active OsRacB. A, The structure of over-expression vector construction for OsRacB. B, Total RNA was extracted from the leaves of OsRacB over-expression lines (R1, R2, R3, R4, and R6). Lane 1 is the vector control. Hybridization and northern analysis was carried out as described in Fig. 2.

to that of the HR-like lesion size in vector plants (Fig. 5A). The latter result suggests that the level of R-gene mediated disease resistance is compromised due to OsRacB over-expression. These results demonstrated that OsRacB over-expression results in increased susceptibility to blast pathogens. Therefore, the gene expression and transgenic data on OsRacB suggest

The question arises how does the OsRacB function in regulating the signaling pathway for susceptibility to the blast fungus in rice? A plausible answer is that like other plant ROPs [9, 16,21], OsRacB might be targeted to the plasma membrane (PM) in order to establish an interaction with a PM-localized receptor for transmitting extracellular signals to its downstream signaling pathway(s). The C-terminal HVR of OsRacB indeed contains a typical prenylation signal “CAAX motif” (amino acid: CSIL), which has been shown to be involved in the localization of HvRacB at the PM [21]. To examine as to whether OsRacB is localized at the PM, we employed three different approaches using the same Pro35:GFP:OsRacB chimeric construct. In a first approach, the fusion protein was transiently expressed in onion epidermal cells after biolistic transformation (Fig. 6A). We observed the localization of GFP fusion proteins 24 h after transformation under blue light by using a fluorescence microscope (Fig. 6B). Expression of the GFP protein alone resulted in green fluorescence of the entire cytoplasm and nucleoplasm. In contrast, we always observed GFP::OsRacB fluorescence exclusively at the cell periphery, indicating association of OsRacB with PM. In a second approach, we

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Fig. 5. Over-expression transgenic lines of OsRacB show increased susceptibility to a virulent (compatible) race of rice blast fungus. A, Disease responses of transgenic plants after infection with virulent and avirulent (incompatible) races of Magnaporthe grisea. Photographs were taken 4 and 6 days after inoculation (race 007 and KI1117, respectively). Vector control, and the R1, R2, R3, R4, and R6 independent transgenic rice of OsRacB over-expression lines are marked. Race of the blast fungus; virulent (007, KI1117), avirulent (S102). B, The histograms show disease severity (virulent 007 and KI1117), evaluated at 4 days post-inoculation: race 007, and 6 days post-inoculation: race KI1117, according to the six-point disease index scale [13]. The bars are represented by standard error.

prepared Arabidopsis protoplast from T2 homozygous transgenic plant and examined intracellular localization of OsRacB (see Materials and methods). Microscopic analyses clearly indicated that GFP:OsRacB fluorescence is localized at the PM (Fig. 7). Together, these results demonstrate that OsRacB is localized at the PM, and therefore, OsRacB perhaps primarily functions at the PM. Even if we have not conducted mutation analysis of the prenylation signal (CSIL at C-terminus of OsRacB) of OsRacB, its barley ortholog HvRacB has been studied in this regard [21]. Deletion analysis of CSIL of HvRacB has demonstrated that CSIL is required for both the specific localization at the PM and the accurate function in barley powdery mildew fungus interaction [21]. As HVR region and CAAX motif are 100% identical between OsRacB and HvRacB, it is reasonable to suggest that OsRacB localization at the PM is necessary for its function. 3. Conclusion This study develops a concept and provides a number of evidence, suggesting that OsRacB is a negative regulator of a

basal disease resistance pathway(s). We also show that OsRacB is localized at the PM, which is most likely required for OsRacB function. Though OsRacB response seems to be affected by incompatible rice-blast pathogen interaction, further experimentation is required to establish association of OsRacB with R-gene mediated disease resistance pathway(s). 4. Materials and methods 4.1. Biological materials Rice (O. sativa L. japonica-type cv. Nipponbare) was grown under white fluorescent light (wavelength 390–500 nm, 150 μmol.m−2.s−1, 12 h photoperiod) at 25 °C and 70% relative humidity as described previously [2–4]. Fungal pathogen (blast fungus, M. grisea races KI1117) was obtained from the Rural Development Administration (RDA), Korea; KI indicates Korean races infecting indica. The M. grisea race 007 and S102 were kind gifts from Dr. Nagao Hayashi (National Institute of Agrobiological Sciences, Tsukuba, Japan).

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Fig. 6. Plasma membrane localization of the GFP::OsRacB protein. A, Construction of GFP-tagged OsRacB fusion protein. B, GFP-tagged OsRacB constructs were introduced into onion cell by particle bombardment. The onion cells were incubated at 25 °C for 24 h and transient expression was observed using fluorescence microscopy according to the Material and methods. OsRacB is localized to the plasma membrane in the bombarded cells.

4.2. Chemicals CN and JA were purchased from Sigma (St. Louis, MO, USA), whereas EN was obtained from BIOMOL Research Laboratories (PA, USA). All other chemicals used in this study were of analytical grade. Stock solutions were prepared as reported previously [2–4] ABA, chitosan, ET, JA, and SA were used for treatments at 100 μM concentrations. 4.3. In vitro system The middle portions of fully expanded leaves (approximately 2 cm long segments cut with sterile scissor) from twoweek-old seedlings were used for all in vitro treatments under continuous light (150 μmol.m−2.s−1) [2–4] Leaf segments were sampled at the times indicated, and immediately frozen at –80 °C. Leaf segments floated on Milli Q (MQ) water served as an appropriate control (also called wounding by cut) in these experiments. 4.4. cDNA library construction, screening and sequence analysis The poly (A)+ RNA (5 μg) was prepared from total RNA isolated from rice PBH, which served as the template for cDNA synthesis and construction of a Uni-ZAP XR vector (Stratagene, La Jolla, CA, USA). The amplified library con-

tained 4 × 109 pfu/ml. The cDNA library was screened using 3′-untranslated region (3′-UTR) of barley HvRacB as a probe following the manufacturer’s recommendation. Autoradiography was carried out overnight with X-ray films (Kodak, Tokyo, Japan) using two intensifying screens at –80 °C. Positive phage clones were selected, and phagemids (pBluescript SK-) were rescued through in vivo excision protocol (Stratagene). Both strands of the recombinant phagemids of positive clones were sequenced using a dye-terminator cycle sequencing kit, and an automated capillary DNA sequencer (Genetic Analyzer ABI 310, PE Applied Biosystems). All sequencing data were analyzed using Genetyx software (SDC Software Development, Tokyo, Japan). Homology of nucleotide and amino acid sequence was analyzed using Genetyx and CLUSTAL-W programs against sequences in the GenBank and EMBL DNA database. 4.5. Northern analysis Total RNA was isolated from rice seedling leaves using the TRIzol(R) reagent (Gibco/BRL, USA), and blotted onto a nylon membrane (Hybond-N+, Amersham). Northern analyses were carried out as described previously [2–4]. The membrane was hybridized with a [α-32P]dCTP-labeled (Megaprime DNA labeling system, Amersham) OsRacB PCR amplified probe (the forward primer of OsRacB-F: 5′-AAAGGGGGGCGTGCTC CA-3′ and the reverse primer OsRacB-R: 5′-AGGAAATAC CATCTAGG-3′; product size 258 bp). Membranes were

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Fig. 7. Subcelluclar localization of OsRacB in the Arabidopsis protoplasts. The OsRacB coding region was fused into the GFP frame of the pK7WGF2 and transformed into the Arabidopsis (Col-0). Homozygote T2 transgenic lines were harvested and protoplasts were prepared according to the protocol described in Materials and methods. Localization was observed in the plasma membrane with GFP and FITC filters using the protoplasts from the transgenic plants. C indicates cytoplasm, and PM indicates plasma membrane.

washed with 2 × SSC and 0.1% SDS at 65 °C for 1 h, and exposed to an X-ray film (Kodak) using two intensifying screens for 2 days at –80 °C. 4.6. Vector constructions To construct the over-expression vector, the complete openreading frame (ORF) of OsRacB cDNA (1066 bp) in the pBluescript SK- vector was digested with XbaI and KpnI, and inserted downstream of the Cauliflower mosaic virus (CaMV) 35S promoter into the multicloning sites XbaI and KpnI of the pCambia1300 binary vector. Complete ORF of the OsRacB cDNA was also subcloned into pK7WGF2 vector (VIB, Belgium) for transient localization assay. The cDNA were cloned using Gateway DNA recombination system (Invitrogen, USA). The full ORF of OsRacB was amplified with PCR primers having adapted attB sequence with two step PCR amplifications. First PCR primers used for OsRacB amplification are 5′-AAAAAAG CAGGCTGGATGAGCGCGTCCAGGTTCATA-3′ and 5′AGAAAGCTGGGTTCACAAAATGGAGCACGCCCCC-3′ (attB sequences are indicated as bold, the rest sequences are OsRacB ORF including start codon underlined). The PCR products were used as templates for the second PCR reaction to completely add attB sequences to the first PCR product DNAs. (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′, 5′– GGGGACCACTTTGTACAAGAAAGCTGGGT-3′, newly

added attB sequences are underlined and previously added attB sequences are bold). Amplified PCR products by two-step PCR were recombined with BP clonase (Invitrogen) into the pDONR vector yielding an entry clone and a ccdB fragment by-product. The entry clone containing OsRacB recombines with a destination vector with LR clonase (Invitrogen) to generate GFP:OsRacB chimeric construct according to the procedures described by the company (Invitrogen). This PK7WGF2 vector with OsRacB ORF was used for localization study in onion cells and Arabidopsis. 4.7. Generation of transgenic rice with OsRacB The japonica rice cv. Nipponbare was transformed with Agrobacterium tumefaciens strain LBA4404 harboring overexpression binary vector of OsRacB (Pro35S:OsRacB), following standard procedure [11] with some modification. Transformed calli were selected on the gradually increased level of hygromycin (30 and 60 mg hygromycin/l) media during in vitro culture. After shooting and rooting from the transformed and selected callus, seedlings were acclimated for 3–4 days in water, and then, they were grown in soil in a greenhouse. Seeds of T1 lines were analyzed for hygromycin resistance, and the hygromycin-resistant seedlings were used for functional analysis and T2 homozygote selections.

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4.8. Pathogen, inoculum production, and inoculation methods Rice plants were grown in a transparent glass bottle (height; 30 cm, diameter; 11 cm) for pathogen inoculation and disease evaluation. Each bottle carried 200 ml of 1/2 MS media (15 g sucrose, 2.15 g Murashige and Skoog basal salt mixture (MS salt) and 3 g Phyta gel/l) and ca. 10 sterilized (1% Clorax and washed three times with sterilized water) rice seeds were placed on the surface of the media. Plants at the 3–4th leaf stages, after ten days in the growth chamber at 28 °C, were inoculated with the prepared M. grisea spores (5 × 105 conidia ml−1) by spraying with an air compressor. Conidial spore suspension was prepared from ten-day-old colonies (M. grisea races 007, KI1117, and S102) that were grown on an oatmeal agar medium (50 g oatmeal per liter) with 250 ppm Tween 20 in distilled water. Bottles carried inoculated rice plants were incubated at 25 °C for 24 h in the dark, and transferred to 28 °C under the previous normal growth condition. Disease development was evaluated 4 to 6 days after inoculation (Race 007, and S102; 4 days post-inoculation, and KI1117; 6 days post-inoculation). Inoculated leaves were collected at desired time period of post-inoculation. Leaves collected before inoculation served as a control sample (0 h). All collected samples were immediately frozen in liquid nitrogen, and stored at –80 °C. Homozygote T2 transgenic lines were screened for 1 week on the selection medium (60 mg hygromycin/l MS). The transgenic plants were moved to MS medium, and were inoculated as described above. Disease severity was scored after 4 and 6 days by inoculation with rice blast fungus race 007, KI1117, and S102 according to the six-point disease index scale [13]. 4.9. RT-PCR Total RNA was isolated from rice tissues using the QIAGEN RNeasy Plant Mini Kit (QIAGEN Cat. No. 74904, Maryland, U.S.A.). Total RNA samples were DNase-treated with an RNase-free DNase (Stratagene, La Jolla, CA, U.S.A.) prior to RT-PCR. First-strand cDNA was synthesized in a 50 μL reaction mixture with a StartaScriptTM RT-PCR Kit (Stratagene, U.S.A.) according to the protocol provided by the manufacturer, using 10 μg total RNA isolated from rice leaves 2, 3, and 4 days after inoculation with M. grisea race 007 or S102 (see section 4.8.). The 50 μl reaction mixture (in 1 X buffer recommended by the manufacturer of the polymerase) contained 1.0 μl of the first-strand cDNA from above, 200 mM dNTPs, 10 pmol of each specific primer set [(OsRacB-F was 5′-GTGCTGTTCCTATCACCACTGCT-3′ and the reverse primer OsRacB-R was 5′-GCAGCTGCCAGTAAACTAGGAT3′; 330 bp), and (OsPR1b-F was 5′-CAGCAACCGGAA CAACCTT-3′ and the reverse primer OsPR1b-R was 5′GTACGTACGCCCGTGTGTATAA-3′; 330 bp)], and 0.5 U of taq polymerase (TaKaRa Ex Taq Hot Start Version, TaKaRa Shuzo Co. Ltd., Shiga, Japan). Thermal-cycling parameters were as follows: after an initial denaturation at 97 °C for 5 min, samples were subjected to a cycling regime of 30 cycles

at 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min. At the end of the final cycle, an additional extension step was carried out for 10 min at 72 °C (TaKaRa PCR Thermal Cycle Dice, Model TP600, Tokyo, Japan). After completion of the PCR, the total reaction mixture was mixed with 2.0 μl of 10X loading buffer, vortexed, and 10 μl was loaded into wells of a 1.6% agarose (Agarose ME, Cat. No. 50013 R, Iwai Chemicals, Tokyo, Japan) gel, followed by electrophoreses for ca. 30 min at 100 V in 1X TAE buffer, using a Mupid-ex electrophoresis system (ADVANCE, Tokyo, Japan). The gels were stained (20 μl of 50 mg/ml Ethidium bromide in 100 ml 1X TAE buffer) for ca. 10 min, and the stained bands were visualized using an UV-transilluminator (ATTO, Tokyo, Japan). The intensity of each band (area) was calculated using the ATTO lane and spot analyzer ver 6.0 (ATTO, Japan). 4.10. Localization and visualization analysis GFP fusion protein was constructed with a full length OsRacB clone with the GFP N-terminus in a CaMV 35S promoter. For transient localization assay, the Pro35S:GFP:OsRacB construct DNA was purified as plasmids using Qiagen plasmid miniprep kit (Qiagen, USA). Plasmid DNA was mixed with tungsten particles and particles coated with Pro35S:GFP:RacB construct plasmids were transformed into onion cells by particle bombardment [7]. Leaf samples were placed on MS media in the Petri plate. The Pro35S:GFP:OsRacB chimeric constructs were introduced into Arabidopsis (Col-0) by spraying method. Agrobacterium was cultured for 2 days in 25 ml LB liquid media until the OD600 reached 0.8. Agrobacterium was harvested by centrifugation with 3,000 rpm, and the pellet was resuspended with the transformation liquid solution (5% sucrose, 0.05% Silwet L-77) to make the final concentration of OD600 = 1. The conidial suspension with OD600 = 1 of Agrobacterium suspension was sprayed using an air-brush onto the flower part of the plants before flowers begin to bloom. The spraying was carried out 2 times with 2 days intervals. T2 generation of transgenic homozygote plants expressing GFP images were identified as above. Homozygote T2 transgenic Arabidopsis plants were used for protoplast preparation. Leaves were gently prepared by razor blade from the 2/3week-old transgenic lines, and they were cut into the small pieces (0.5–1 g, 5–10 mm2). Cut pieces were transferred to enzyme solution (0.5 M mannitol, 2% cellulose-RS, YAKULT HONSHA, Japan, 0.05% pectolyase Y-23, KYOWA, Japan, pH 5.8) and vacuum infiltrated for 2 min at 15 mmHg, followed by incubation in the dark at 32 °C for 1-2 h with gentle agitation (50-75 rpm). Digestion solution was filtered through 100 mm mesh (Sigma, USA) for the protoplast separation. The epidermal cells and protoplasts expressing GFP images were identified using an Olympus microscope with epifluorescence using GFP-optimized ND filter sets (Olympus, Japan). Analyses were conducted under bright light, GFP filter (excitation; 450–500 nm, beamsplitter; FT510 nm, excitation; BP515-565 nm), and FITC filter (excitation; 450–490 nm, beamsplitter; FT510 nm, emission; LP515 nm). Digital images were col-

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lected using Olympus IX70 fluorescence microscope (Olympus) with I.CAMSCOPE digital camera (Sometech, Korea) and software (MicroFire, USA). Images were further processed with Photoshop 5.0 software (Adobe). Acknowledgements This research was supported by a grant from the Plant Signaling Network Research Center, the Korea Science and Engineering Foundation, and the Crop Functional Genomics Center of the 21st Century Frontier Research Program (Grant No. CG1412). References [1]

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