Identification of a cluster of PR4-like genes involved in stress responses in rice

Journal of Plant Physiology 168 (2011) 2212–2224

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Identification of a cluster of PR4-like genes involved in stress responses in rice Nili Wang a , Benze Xiao b , Lizhong Xiong a,∗ a b

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

a r t i c l e

i n f o

Article history: Received 17 March 2011 Received in revised form 24 June 2011 Accepted 6 July 2011 Keywords: Abiotic stress Cross-talk Phytohormone PR protein RNase activity

a b s t r a c t PR4 proteins constitute a pathogenesis-related (PR) protein family with a conserved BARWIN domain. In this study, we analyzed PR4-homologous genes in rice (Oryza sativa L.) and identified five putative PR4 genes designated as OsPR4a−e. The five PR4 genes are located in tandem on chromosome 11 and constitute a gene cluster with high sequence similarity to each other. The OsPR4 proteins have high sequence similarity to reported PR4 proteins from monocotyledonous species and are predicted to be class II PR4 proteins. Distinct diversification of plant PR4 proteins exists between monocotyledonous and dicotyledonous plants. Except for OsPR4e, which was not detected with any transcript, the other four OsPR4 genes showed diverse temporal–spatial expression patterns, and their expressions are responsive to Magnaporthe grisea infection. Interestingly, the OsPR4 genes are also responsive to abiotic stresses. Their expression levels were strongly induced by at least one of the stress treatments including drought, salt, cold, wounding, heat shock, and ultraviolet. The transcript levels of OsPR4 genes were also induced by some phytohormones such as abscisic acid and jasmonic acid. Transgenic rice with overexpression of OsPR4a showed enhanced tolerance to drought at both seedling and reproductive stages. We conclude that rice PR4 genes are also involved in abiotic stress responses and tolerance in addition to their responsiveness to pathogen attacks. © 2011 Elsevier GmbH. All rights reserved.

Introduction Plants are frequently exposed to various abiotic stresses and sometimes together with biotic stresses in their natural environment. To survive such adverse conditions, they have evolved intricate mechanisms to respond and adapt to abiotic and biotic stresses (Fujita et al., 2006). When a plant is infected by pathogens, a large number of genes that are involved in transcriptional regulation, signal transduction, various metabolic activities, and defense responses are activated (De Vos et al., 2005). Most of the defense-related proteins are called pathogenesis-related (PR) proteins. The PR proteins reported so far can be grouped into 17 independent families (van Loon et al., 2006). The plant PR4 family consists of two classes of proteins. Class I PR4 proteins contain a conserved N-terminal cysteine-rich chitin-binding domain (or hevein domain), which corresponds to an antifungal protein from rubber tree latex (Hevea brasiliensis) (Broekaert et al., 1990). In contrast, class II PR4 proteins lack the chitin-binding domain. All PR4 proteins have a C-terminal BARWIN domain. This term was derived from a basic barley seed protein BARWIN, which contains six cysteine residues that can form three

∗ Corresponding author. Tel.: +86 27 87281536; fax: +86 27 87287092. E-mail address: [email protected] (L. Xiong). 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.07.013

disulfide bridges (Cys31–Cys63, Cys52–Cys86, and Cys66–Cys123) and has the ability to bind saccharides (Ludvigsen and Poulsen, 1992b). The secondary structure of this protein has been well defined and three-dimensional structure analysis shows that it consists mainly of a large four-stranded antiparallel beta sheet, a small parallel beta sheet, and three small alpha helices (Ludvigsen and Poulsen, 1992a,b). Most PR4 proteins characterized from different plant species have an N-terminal signal peptide, and some of them also have a C-terminal extension, which is considered a necessity for the proteins to be targeted to vacuoles (Neuhaus et al., 1991). The PR4 protein genes identified for the first time were named win1 and win-2, which are tandemly located on the potato genome and encode two wound-inducible proteins with high homology to each other (Stanford et al., 1989). Since then, a number of PR4 protein genes have been identified from different plant species such as tomato, tobacco, Arabidopsis, Chinese cabbage, wheat, Capsicum chinense, and maize (Linthorst et al., 1991; Caruso et al., 1993; Potter et al., 1993; Bravo et al., 2003; Park et al., 2005; Guevara-Morato et al., 2010). In monocotyledon plants, the maize PR4 gene ZmPR4 was induced by wounding and by treatment with abscisic acid (ABA) or methyl jasmonate, and in situ hybridization analysis showed that accumulation of ZmPR4 mRNA occurred in the cells that confronted pathogen first (Bravo et al., 2003). One wheat PR4 protein Wheatwin1 was proposed to have RNase activity, and both native and recombinant PR4 proteins could hydrolyze RNA from wheat

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coleoptiles and have antifungal activity (Caporale et al., 2004). The RNA-binding site and its interaction with 5 -ADP was later characterized (Bertini et al., 2009). The RNase activity of this protein on endogenous RNA of host cells was proposed as the key for the antifungal activity (Bertini et al., 2009). The recombinant LrPR4 protein expressed in Escherichia coli showed activity toward hydrolyzing RNA from Lycoris radiata bulbs (Li et al., 2010). Another five new genes (wPR4-e, wPR4f-b, wPR4f-a, wPR4f-c, and wPR4g) belonging to the PR4 family have been identified. The wPR4e and wPR4f subfamily genes are induced by Fusarium culmorum infection, and analysis of the 5 untranslated region (UTR) of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements, further suggesting that this gene family may be responsive to various stress conditions (Bertini et al., 2006). Rice is an important food crop and a model species for monocotyledonous plants, but our understanding of PR4 proteins in this species is relatively limited. The first rice PR4 gene, OsPR4, was reported to be differentially induced in response to compatible and incompatible host–pathogen infections (Agrawal et al., 2003). This gene is induced by jasmonic acid (JA), ABA, cantharidin (CN), endothall (EN), and okadaic acid (OA) treatments, but not by wounding, salicylic acid (SA), ethylene (ET), or hydrogen dioxide (H2 O2 ) treatments (Agrawal et al., 2003). The second rice PR4 gene OsPR-4b was reported for the antifungal activity of recombinant protein of OsPR-4b against Rhizoctonia solani in vitro (Zhu et al., 2006). In this study, we identified all putative PR4 genes (five members named OsPR4a–e) in the rice genome and analyzed the expression profiles of this family in different tissues and organs of rice. We also investigated the expression profiles of the OsPR4 genes under various treatments of stresses or chemicals to determine if this family is also involved in abiotic stress responses. The results suggest that OsPR4 genes are strongly induced by various abiotic stresses, and transgenic rice plants overexpressing OsPR4a exhibit significantly improved tolerance to drought. These results provide direct evidence of the involvement of the PR4 family in abiotic stress tolerance in addition to its well-known antifungal function in plants.

Materials and methods Identification of rice PR4 genes Using the BLASTP program, the reported sequences of BARWIN protein (BARW HORVU, accession no. P28814) from barley and pathogenesis-related protein 4 (PR4A TOBAC, accession no. P29062) from tobacco were used as queries to search the rice annotation database at The Institute for Genomic Research (TIGR) (http://www.tigr.org/) and the KnowledgeBased Oryza Molecular Biological Encyclopedia (KOME) (http://cdna01.dna.affrc.go.jp/cDNA/). All putative sequences with an E-value < 0.01 were collected. The transcript sequences were further checked by performing BLASTN searches against the rice (Oryza sativa) EST database. BLASTN search against rice genome sequence (http://www.tigr.org/) was used to determine the chromosome locations of putative OsPR4 genes. Gene structures of the OsPR4 genes were determined by comparing full-length cDNAs or expressed sequence tags (ESTs) with genomic DNA sequences obtained from the TIGR database. Schematic diagram of gene structure was drawn using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). Conserved protein domain analysis was done by scanning protein sequences against the profile entries in the Pfam and EMBL-EBI databases (http://pfam.sanger.ac.uk/; http://www.ebi.ac.uk/) and their physical and chemical characteristics were predicted by ExPASy Proteomics tools (http://expasy.org/tools/).

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Sequence alignment and phylogenetic analysis Twenty-six putative PR4 protein sequences were used for phylogenetic analysis and motif prediction, including all five members from rice, one from Arabidopsis, and 20 from other flowering plants (HbHEV1 from Hevea brasiliensis; EwQ8H950 from Eutrema wasabi; StWIN1, StWIN2 from Solanum tuberosum; SnPR4 from Sambucus nigra; TaWheatwin1, TaWheatwin2 from Triticum aestivum; NtPR4A, NtPR4B, NtCBP20 from Nicotiana tabacum; DbPRP-4-1 from Dioscorea bulbifera; PtA0SWV6 from Populus tremula; CcPR4b from Capsicum chinense; CaCbp from Capsicum annuum; VvPR4a from Vitis vinifera; SlP2 from Solanum lycopersicum; PsPRP4A from Pisum sativum; HvBarwin, HvP93180 from Hordeum vulgare; ZmPR4 from Zea mays). Multiple sequence alignment of PR4 proteins was performed with CLUSTALX (Version 1.83) and Genedoc. The phylogenetic analysis was performed by the program MrBayers version 3.0 (Ronquist and Huelsenbeck, 2003). A total of 400,001 generations were performed with Markov chain and parameter values of trees were sampled every 100 generations. The first 100 sampled trees were discarded. The major consensus tree was deduced from the remaining 3901 sampled trees. The tree was edited with TreeView 1.5. To predict putative cis-acting regulatory DNA elements (ciselements) in the promoters of the OsPR4 genes, 1000-bp regions upstream of the 5 end of the full-length cDNAs or the predicted coding sequence (CDS) were extracted from genomic sequences and subjected to cis-element search in PLACE 26.0 (http://www.dna.affrc.go.jp/PLACE/). Plant materials and stress treatments Seeds of rice variety Zhonghua 11 (O. sativa L. ssp. japonica) were germinated in plates with water. Plants were grown in a greenhouse with a 14-h light/10-h dark cycle at 28 ◦ C and various tissue and organ samples were collected at seedling and reproductive stages. For transcript level analysis of OsPR4 genes under abiotic stress and chemical treatments, rice plants were planted in plastic pots filled with sandy soil and placed in the greenhouse. Seedlings at the four-leaf stage were prepared for different treatments. Drought stress was induced by withholding water. Plant leaves were sampled before treatment and every day at 11:00 am starting from the day when the plants showed slight leaf-rolling (the leaf relative water content [RWC] was 90–91%). Plants were watered again when the leaves became irreversibly rolled (leaf RWC was 75–78%) in the morning and sampled at 12 h after re-watering. For salt stress, the seedlings were irrigated with 200 mM NaCl solution and sampled at 0, 2, 6, 12, 24, and 36 h after salt treatment. For cold and heat shock stress, seedlings were transferred to growth chambers of 4 ◦ C and 42 ◦ C, respectively. For cold treatment, samples were collected at 0, 1, 3, 6, and 12 h after cold treatment. For heat shock treatment, samples were collected at 0, 1, 3, 6, and 12 h after heat treatment. To detect whether OsPR4s plays a role in the recovery process after heat shock treatment, the rest of the seedlings were returned to the greenhouse with a 14-h light/10-h dark cycle at 28 ◦ C for recovery. After 3 d of recovery, samples were collected. For ultraviolet (UV) light stress, seedlings were moved to a hood with UV light (emission peak at 254 nm, 1100 mW cm2 at plant level [TUV30W, Philips]) and sampled at 0, 6, and 12 h during treatment. To detect whether OsPR4s has a role in the recovery process after UV treatment, the rest of the seedlings were moved to the greenhouse with a 14-h light/10-h dark cycle at 28 ◦ C for recovery and sampled after 24 h. For wounding treatment, rice leaves were pierced with pointed forceps and sampled at 0, 1, 3, 6, 12, and 24 h after treatment. Hormone treatments were performed by spraying leaves evenly with brassinosteroid (BR, 10 ␮M), phytokinin (KT, 450 ␮M), gibberellin

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Table 1 Basic information of OsPR4 family genes. Gene name

Previous name

TIGRa

BACb

cDNA accession no.

Exon no.

Intron no.

Protein length

OsPR4a OsPR4b OsPR4c OsPR4d OsPR4e

OsPR4 OsPR-4b

LOC LOC LOC LOC LOC

AC137744 AC137744 AC137744 AC146334 AC146334

AK121127 AK063234 AK121059 AK243507 NA

2 2 2 2 2

2 1 1 2 1

146 152 150 158 102

a b

Os11g37970 Os11g37960 Os11g37950 Os11g37940 Os11g37930

Accession number of OsPR-4 in TIGR database. Accession number of bacterial artificial chromosome clone of rice.

Fig. 1. Genomic organization of OsPR4 family in rice. (A) Chromosomal location of OsPR4 genes. Arrow indicates the direction of the transcription. (B) Exon and intron (illustrated by rectangles and lines, respectively) organization pattern. The pattern is conserved for the PR4 genes in maize (ZmPR4) and Arabidopsis (AtHEL). The UTR regions and BARWIN domains are indicated in white and black, respectively.

(GA, 100 ␮M), ABA (100 ␮M), indole-3-acetic acid (IAA, 60 ␮M), jasmonic acid (JA, 1 mM), ethephon (ETH, 300 ␮M), SA (1 mM), or H2 O2 (200 ␮M) and then sampled at 0, 0.5, 1, 3, 6, and 12 h. For drought testing of overexpression transgenic lines, seeds of T1 overexpression lines were germinated on 1/2 Murashige and Skoog medium with 50 mg/L hygromycin. The vigorously germinated seeds were selected for the experiments. For drought testing at the seedling stage, overexpression and control plants were grown in the same pot. Water supply was halted when plants were grown to the four-leaf stage. After severe drought stress (all leaves wilted and leave RWC was 75–78%) and recovery, the plants were photographed and the survival rate was recorded. For drought stress testing at the reproductive stage, the overexpression and control plants were grown in a refined paddy field facilitated with a movable rain-off shelter. Drought stress was applied at the panicle development stage, which allowed severe drought stress at the flowering stage under this experimental condition, and relative grain yield (ratio of the yield under stress to the yield under nonstress condition) was used as a major criterion for drought resistance evaluation as used in our previous report (Xiao et al., 2007). To examine the influence of pathogen infection on OsPR4 gene expression, seedlings of Minghui 63 (an elite restorer line for indica hybrid rice) and Zhenshan 97 (an elite maintainer line for indica hybrid rice) at the three- to four-leaf stage were inoculated with Magnaporthe grisea isolate 91-17-2 (kindly provided by Professor Peng YL from China Agricultural University) by the spraying method (Chen et al., 2003). Mock-inoculated plants under the same conditions were used as controls.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNAs of collected samples were extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Total RNA was treated with DNase I (Invitrogen) at 37 ◦ C for 15 min to degrade possibly contaminated residual genomic DNA. SuperScript® III reverse transcriptase (Invitrogen) was used to synthesize first-strand cDNA according to the supplier’s protocol. Relative quantification of gene expression was performed by real-time RT-PCR on an ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA). Four pairs of specific primers (PR4aF: 5 -CAACTGGGACCTGAACAAAGTG-3 and PR4aR: 5 -GCGCCAAGACAACGGTTT-3 for OsPR4a; PR4bF: 5 -AGTATGGAT GGACCGCCTTCT-3 and PR4bR: 5 -GACATTTGCCACATGCATCCT3 for OsPR4b; PR4cF: 5 -TCGTGGCGTCAGAAGTATGG-3 and PR4cR: 5 -AGCCTGACCCCTAGGACCAA-3 for OsPR4c; PR4dF: 5 -CTGTGTGGGCCTTGTTGCT-3 and PR4dR: 5 -CTCACGCCG AATGCTTGTT-3 for OsPR4d) for real-time RT-PCR were designed using Primer Express Version 2.0 (Applied Biosystems) under default parameters on the basis of the cDNA sequence of OsPR4a, OsPR4b, OsPR4c, and OsPR4d. The expression level of rice Actin1 gene (accession no. X16280) was used as the internal control with primers 5 -TGGCATCTCTCAGCACATTCC-3 and 5 TGCACAATGGATGGGTCAGA-3 . Real-time RT-PCR was performed in an optical 96-well plate. Each reaction contained 6 ␮L cDNA diluted samples (1/20 of the first-strand cDNA obtained from 2 ␮g total RNA) as template, 200 nM of each gene-specific primer and 12.5 ␮L SYBR® Premix Ex TaqTM (Perfect Real Time) (TaKaRa,

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Fig. 2. Protein sequence alignment of PR4. Class I PR4 proteins from Arabidopsis (ATHEL), Hevea brasiliensis (HbHEV1), Solanum tuberosum, (StWIN1), and Nicotiana tabacum (NtCBP20) and class II PR4 proteins from Nicotiana tabacum (NtPR4A), Zea mays (ZmPR4), Hordeum vulgare (HvBarwin), and Triticum aestivum (TaWheatwin1) were used for sequence alignment with the deduced amino acid sequences of OsPR4 genes. The signal peptide, hevein domain, hinge, BARWIN domain, and vacuolar signal are marked. Arrow indicates the highly probable cleavage sites. Asterisks indicate the six cysteine residues conserved in the BARWIN domain. A triangle indicates the conserved position (valine) of the intron.

Tokyo, Japan), and 0.5 ␮L ROX Reference Dye II (TaKaRa) in a final volume of 25 ␮L. PCR was performed following these steps: denaturing at 95 ◦ C for 10 min, 40–45 amplification cycles at 95 ◦ C for 34 s, annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 1 min. Three repeats were analyzed for each gene. After the PCR procedure, a melting curve was plotted under the following conditions: 95 ◦ C for 15 s, 60 ◦ C for 20 s, and 95 ◦ C for 15 s. The

relative expression levels were identified as described previously (Livak and Schmittgen, 2001). Plasmid construction and rice transformation To generate the OsPR4a overexpression construct, the cDNA clone (EI#77-D14), containing the full-length cDNA of OsPR4a

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Fig. 3. Phylogenetic tree of PR4 proteins from rice, Arabidopsis, and other plants. OsPR4a-e from Oryza sativa nippobare; AtHEL from Arabidopsis thanliana; HbHEV1 from Hevea brasiliensis; EwQ8H950 from Eutrema wasabi; StWIN1 and StWIN2 from Solanum tuberosum; SnPR4 from Sambucus nigra; TaWheatwin1, TaWheatwin2 from Triticum aestivum; NtPR4A, NtPR4B, and NtCBP20 from Nicotiana tabacum; DbPRP-4-1 from Dioscorea bulbifera; PtA0SWV6 from Populus tremula; CcPR4b from Capsicum chinense; CaCbp from Capsicum annuum; VvPR4a from Vitis vinifera; SlP2 from Solanum lycopersicum; PsPRP4A from Pisum sativum; HvBarwin and HvP93180 from Hordeum vulgare; and ZmPR4 from Zea mays. The collected protein sequences can be classified into three subgroups, S1, S2, and S3.

was chosen from a normalized cDNA library of Minghui 63 (http://redb.ncpgr.cn/modules/redbtools/) (Chu et al., 2003). Plasmid was digested by KpnI and BamHI and ligated into the destination vector pCAMBIA1301H digested with KpnI and BamHI, thus allowing the genes to be driven by a drought-inducible promoter of the gene OsLEA3-1 (Xiao et al., 2007). This construct was introduced into japonica rice Zhonghua 11 by Agrobacteriummediated transformation (Lin and Zhang, 2005). Results Identification and sequence analysis of PR4-like genes in rice To identify all putative genes of the PR4 family in the rice genome, two reported proteins, BARWIN from barley and PR4A from tobacco, were used as queries to search against public databases of rice. In addition to the reported OsPR4 (LOC Os11g37970, renamed OsPR4a here) and OsPR4b (LOC Os11g37960), three other putative PR4-like genes, designated as OsPR4c (LOC Os11g37950), OsPR4d (LOC Os11g37940), and OsPR4e (LOC Os11g37930), were identified in the rice genome

(Table 1). Except for OsPR4e, the other four OsPR4 genes were supported with full-length cDNAs and ESTs. By BLASTN search against the rice genome, we noticed that the five OsPR4 genes were tandem located on chromosome 11 and they formed a gene cluster (Fig. 1A). Pair wise comparisons suggest a high sequence similarity among OsPR4a, OsPR4b, OsPR4c, and OsPR4d, with the lowest sequence identity (60.1%) between OsPR4a and OsPR4d and the highest (83.4%) between OsPR4b and OsPR4c. The OsPR4e shows relatively low sequence identity (about 48%) to other members. Exon–intron structures of the OsPR4 genes were illustrated by comparing the full-length cDNAs or predicted CDS with the genomic sequence (Fig. 1B). The result showed that each gene contains two exons and one intron in the CDS region. Such a genomic organization (CDS region split by one intron) also exists for the PR4-homologous genes in maize and Arabidopsis (Fig. 1B). The sequence corresponding to the conserved BARWIN domain was identified by scanning the OsPR4 protein sequences against Pfam and EMBL-EBI databases. We noticed that the CDSs for the conserved BARWIN domains were interrupted by the intron at a similar position. These results suggest that the OsPR4 family genes share a very similar gene structure and high sequence identity

N. Wang et al. / Journal of Plant Physiology 168 (2011) 2212–2224 Fig. 4. Expression level of OsPR4 genes in various tissues or organs. (A) Expression levels examined by quantitative real-time RT-PCR. Twelve tissues or organs were checked: (1) seed; (2) young root; (3) culm (heading stage); (4) node; (5) hull; (6) secondary branching of inflorescence; (7) anther; (8) pistil; (9) young shoot; (10) pulvinus; (11) flag leaf in the morning; and (12) sheath. (B) Expression profiles of OsPR4 genes in tissues and organs covering a whole life cycle of rice. Data were collected from the Collection of Rice Expression Profiles (CREP) database. 2217

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Fig. 5. Expression level of OsPR4 genes examined by real-time RT-PCR in two rice varieties infected with Magnaporthe grisea isolate 91-17-2. DAI indicates day after inoculation. CK, no infection.

and constitute a young gene family with the members most likely derived from tandem gene duplication. The predicted proteins of the OsPR4 family are also similar in length to amino acids (range, 146–158). Analysis with server (http://www.cbs.dtu.dk/services/SignalP/) SignalP3.0 showed high probability scores of cleavage between the 26th and 27th amino acids of OsPR4d (0.802) and between the 24th and 25th amino acids of OsPR4c (0.945) (Fig. 2). Protein sequence alignments of OsPR4 family and several known class I and class II PR4 proteins from monocotyledonous and dicotyledonous species revealed high similarity of the OsPR4 proteins to the known class II PR4 proteins. They all contain an N-terminal signal peptide and a BARWIN domain but lack the N-terminal cysteine-rich Hevein domain (Fig. 2). However, some variations exist in the OsPR4 family. For example, about 40 amino acids of the BARWIN domain are missed in OsPR4e. Such an incomplete BARWIN domain may indicate that OsPR4e may be a nonfunctional gene. In wheat, two PR4 genes, wPR4f-b and wPR4f-c, are also predicted to produce non-functional chimeric proteins (Bertini et al., 2006). OsPR4d contains a C-terminal extension that is present in the class I of the PR4 family and was thought to be required for targeting the proteins to vacuoles (Neuhaus et al., 1991).

Phylogenetic analysis of PR4 family To investigate the evolutional relationship of PR4 genes in plants, we performed phylogenetic analyses of 26 putative PR4 protein sequences, including all five members from rice, one from Arabidopsis, and 20 from other flowering plants (listed in ‘Materials and methods’). Based on the phylogenetic tree constructed, the collected proteins can be classified into three subgroups, S1, S2, and S3 (Fig. 3). Except for OsPR4e, all the PR4 proteins from monocotyledon species (e.g., rice, maize, and wheat) fall into S2, and all the PR4 proteins from dicotyledonous species fall into S1 and S3. This result suggests that the diversification of PR4 family may have occurred mainly after the divergence of dicotyledonous and monocotyledon plants. In addition, we noticed that the proteins in the

S2 and S3 subgroups belong to class II PR4 and those proteins in S1 feature to class I PR4. Expression levels of OsPR4 genes in different tissues and organs The expression levels of the OsPR4 gene family were checked in 12 representative rice tissues and organs, including (1) seed; (2) young root; (3) culm (heading stage); (4) node; (5) hull; (6) secondary branching of inflorescence; (7) anther; (8) pistil; (9) young shoot; (10) pulvinus; (11) flag leaf in the morning; and (12) sheath, by real-time RT-PCR (Fig. 4A). We designed gene-specific PCR primers for the five OsPR4 genes but no specific amplification was obtained for OsPR4e in all samples investigated, which further demonstrated that OsPR4e may be a nonfunctional gene. The relative expression levels of the other four OsPR4 genes varied significantly among the tissues or organs checked. Compared to their own expression level in flag leaf (the expression level was established at 1), OsPR4a, OsPR4b, and OsPR4c showed higher expression in hull, secondary branches of inflorescence, young shoot, pulvinus, and leaf sheath. In addition, OsPR4a, OsPR4b, and OsPR4c all showed weak expression in culm and anther. OsPR4d showed relatively specific expression in young root and young shoot and very weak expression in other tissues/organs. The real-time RT-PCR result was compared to the microarray result of OsPR4 family (Fig. 4B) with the data collected from the Collection of Rice Expression Profiles (CREP) database (http://crep.ncpgr.cn/crepcgi/element multi chronologer view.pl) in which the tissues and organs used for profiling cover a whole life cycle of rice (Wang et al., 2010). Except for a few small discrepancies (e.g., the expression of OsPR4a in hull was weak in the microarray but was high in the real-time RT-PCR analysis), most of the data points from the two methods agree very well. Expression of OsPR4 genes under fungal infection Although the PR4 family has been implicated as PR proteins, the homologous PR4 genes in rice were seldom investigated for their responses to pathogen infection. Therefore, we checked the

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Fig. 6. Expression level of OsPR4 genes under abiotic stresses examined by real-time RT-PCR. DAW, days after water stress (see ‘Materials and methods’ for detail); AR, after recovery.

expression levels of OsPR4 genes on the infection of blast fungus (M. grisea isolate 91-17-2) in two rice varieties, Minghui 63 and Zhenshan 97. The result showed that Zhenshan 97 was moderately susceptible and Minghui 63 was relatively resistant to the fungal infection (data not shown). In both varieties, the expression levels of OsPR4a, OsPR4b, and OsPR4c were induced 1 or 2 d after inoculation. OsPR4d was induced more than 10-fold in Minghui 63 at the second day after inoculation. However, no significant change of this gene was detected in Zhenshan 97 (Fig. 5). Expression of OsPR4 genes under abiotic stresses To answer whether OsPR4 genes are also involved in abiotic stress responses, we checked their expression levels in response

to different stress treatments. The results showed that the OsPR4 genes are indeed responsive to abiotic stresses with diverse expression patterns (Fig. 6). All four genes were induced within 12 h after wounding treatment, and the induction levels of OsPR4c and OsPR4d were especially high. With heat shock treatment, OsPR4a, OsPR4b, and OsPR4c showed a similar pattern with expression levels reduced at the early stage of the stress, peaked at 6 h, and then decreased after recovery for 3 d. In the low-temperature treatment, however, no obvious changes in expression levels were observed for the four OsPR4 genes. OsPR4d was very strongly induced (>300fold of induction at the peak) by UV treatment, which was much stronger than the inductions of the other three genes. OsPR4c was induced by treatment with salt (>20-fold) and H2 O2 (>10-fold), but the other three genes showed no obvious fluctuation with these two

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Fig. 7. Expression level of OsPR4 genes under phytohormone treatments examined by real-time RT-PCR. ABA, abscisic acid (100 ␮M); BR, brassinosteroid (10 ␮M); ETH, ethephon (300 ␮M); GA, gibberellins (100 ␮M); IAA, indole-3-aceticacid (60 ␮M); JA, jasmonic acid (1 mM); KT, phytokinin (450 ␮M); SA, salicylic acid (1 mM).

compounds. OsPR4a, OsPR4b, and OsPR4c were induced to different degrees by drought stress and the induction maintained relatively high even after recovery for 12 h. Taken together, these results suggest that the OsPR4 family genes are responsive to various abiotic stresses with very diverse induction patterns. Expression of OsPR4 genes under treatment with various phytohormones Since the OsPR4 genes are responsive to diverse sources of stress, we further detected their expression levels after treatment with various stress-related phytohormones such as ABA, SA, JA, and

ETH (Fig. 7) because these phytohormones have important roles in plant defenses against both biotic and abiotic stresses through complex signaling cross-talks. The OsPR4 genes were also checked for their responsiveness to other phytohormones such as BR, KT, GA, and IAA (Fig. 7). With ABA treatment, OsPR4a, OsPR4b, OsPR4c, and OsPR4d showed a similar expression pattern: reduced at the first 3 h, and then increasing gradually and peaking at the late stage of the treatment. OsPR4d was strongly induced by JA (>100fold), and it was also induced by SA, IAA, BR, GA, and KT (>4-fold). OsPR4c was induced by IAA, BR, and KT (>4-fold). Expression levels of OsPR4a and OsPR4b were increased after JA treatment but fluctuated slightly under SA treatment.

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Fig. 8. Analysis of the promoters of OsPR4 genes. Putative cis-elements, ABA-responsive element (ABRE), MYB-binding site (MBS), and wounding responsive element (W-box) are indicated within 1000 bp upstream of the 5 end of the full-length cDNAs of OsPR4 genes. Inductions by stress treatments are indicated by checks (D, drought; S, salt; A, ABA; C, cold; W, wounding).

Analysis of the promoters of OsPR4 genes We analyzed the 1000-bp sequence upstream of the 5 end of the full-length cDNAs for the OsPR4 genes to identify putative cis-elements (Fig. 8) and noticed enrichment of a few stressresponsive elements, such as ABA-responsive element (ABRE), MYB-binding site (MBS), and wounding responsive element (Wbox) that have been characterized (Urao et al., 1993; Simpson et al., 2003; Nishiuchi et al., 2004; Nakashima et al., 2006). ABRE exists in the promoters of OsPR4a, OsPR4b, and OsPR4c but not in the promoter of OsPR4d, and this result is consistent with the expression patterns of these genes under ABA treatment. All the OsPR4 genes contain MBS, a putative drought-responsive cis-element, in their promoters. To our surprise, we did not find any putative W-box in the promoter region of OsPR4d, even though this gene was strongly induced by wounding, which implies that the novel woundingresponsive cis-element may exist in the promoter of this gene.

Overexpression of OsPR4a in rice resulted in enhanced tolerance to drought A significant finding of this study is that the OsPR4 genes are responsive to abiotic stresses. Therefore, we further asked whether these genes are actually involved in abiotic stress tolerance. To test this, OsPR4a was overexpressed in japonica rice Zhonghua 11 under the control of a drought-inducible promoter (Fig. 9A). Of 10 independent transgenic plants, three showed overexpression of the transgene according to the real-time RT-PCR result (Fig. 9B), and these transgenic lines were tested for drought resistance. For drought resistance testing at the seedling stage, three positive overexpression lines (H2, H7, and H9) and two negative transgenic (control) lines (H5 and H8) were used. Under normal conditions, no significant difference in morphologic traits was observed. When the water supply ceased, overexpression plants spent more time than the control plants exhibiting obviously stressed phenotypes such as leaf-rolling and wilting. After 7 d of drought stress (all leaves wilted) followed by re-watering, the overexpression lines had a significantly higher survival rate (60–70%) than the negative controls (<25%) (Fig. 9C and D). Drought resistance of the three overexpression lines was also tested at the reproductive stage (2 weeks before flowering). Again, no significant difference between overexpression and control lines was observed under normal growth conditions. However, after drought treatment, the overexpression lines had significantly higher relative yields than the negative

control lines (Fig. 9E). These results suggest that OsPR4a has a significant effect on improving tolerance to drought stress in rice.

Discussion In this study, we identified five putative PR4 genes (OsPR4a−e) that are located in tandem on chromosome 11 and constitute a gene cluster with high sequence similarity to each other. The genomic organization of the OsPR4 genes is interesting. Each OsPR4 gene contained two exons and one intron in the CDS. The intron phase is also conserved (phase 2) in these genes (Fig. 1). After analyzing the CDS and genomic sequence for all OsPR4 genes, we found that the CDS was broken at the same conserved valine amino acid. Interestingly, the Arabidopsis PR4 gene AtHEL and maize PR4 ZmPR4 share the same exon–intron structure with OsPR4 and the CDSs of AtHEL and ZmPR4 are also broken by an intron at the conserved valine site. These results may indicate that the genomic structure and splicing model of PR4 genes are conserved in plants. To cope with continuous exposure to abiotic and biotic stresses in the natural environment, plants have evolved a series of responsive and adaptive mechanisms to survive. Increasing evidence suggests that, in addition to the distinctive signal perception and transduction pathways between biotic and abiotic stress responses, some overlap exist in the responses of plants to the two types of stress, thus forming sophisticated networks. These networks include signaling cross-talks among phytohormones, such as ABA, SA, JA, and ET (Anderson et al., 2004; Mauch-Mani et al., 2005; Jiang et al., 2010), generation of reactive oxygen species (Torres and Dangl, 2005), and a large number of genes that function in both biotic and abiotic stresses, for example, CaCDPK3 (Chung et al., 2004), OsMAPK5 (Xiong and Yang, 2003), and BOS1 (Mengiste et al., 2003). PR proteins have been well known for their roles in defense responses to biotic stress but limited evidence has been reported for their involvement in abiotic stress. In this study, we found that genes in the rice PR4 family are responsive to various abiotic stresses and increasing the expression of one member can actually improve drought resistance. This finding further supports the existence of overlaps in the responsive or adaptive mechanisms between biotic and abiotic stresses. In previous reports, the transcript level of OsPR4a was increased in rice leaves infected with the blast pathogen, M. grisea, and OsPR4b protein showed in vitro antifungal activity against the sheath blight fungus, R. solani (Agrawal et al., 2003; Zhu et al., 2006). Here, we checked the expression levels of all four expressed

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Fig. 9. Improved drought resistance of OsPR4a-overexpression rice. (A) Diagram of the OsLEAP:OsPR4a construct. OsLEAP is a drought-inducible promoter. (B) The expression level of OsPR4a in 10 independent transgenic plants (H1−H10) with wild-type Zhonghua 11 as control. (C) Phenotype of three T1 lines of OsPR4a overexpression (H2, H7, and H9) and two transgenic-negative (H5 and H8, the target gene showed no over-expression) lines under drought stress in seedling stage; i−iii before drought stress, iv−vi, 5 d after recovery. (D) Survival rates (the percentage of the number of surviving plants under the drought stress to the total number of plants) after drought treatment. (E) Relative yield of the OsLEAP:OsPR4a transgenic rice after severe drought stress treatment at panicle development stage. Relative yield is the percentage of the yield under the drought stress to that under normal conditions. The bar is standard error based on 25–30 plants.

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OsPR4 genes in two rice lines with M. grisea infection. The increased expression levels of OsPR4a and OsPR4b in both the susceptible and resistant lines confirmed the previous result. The induction of OsPR4c by the fungal infection indicates that it may also have a positive role in rice antifungal activity. Interestingly, OsPR4d is induced only in the relatively resistant line Minghui 63 but not in the susceptible line Zhenshan 97. This result implies a potential contribution of this gene to the difference of the two lines in blast disease resistance. Most PR genes are responsive to defense response-related phytohormones (such as JA, SA, and ET) and wounding. We noticed that the OsPR4 genes had stronger induction by JA than by SA or ETH treatment and were up-regulated after wounding treatment. These results suggest that wounding could trigger the expression of OsPR4 genes and their possible defense activities may be mainly regulated by the JA signaling pathway. It is well known that ABA is an important “stress” hormone that is involved in many aspects of plant development and adaptive responses to various abiotic stresses such as drought, cold, and salinity. An ABA-dependent signaling pathway has an important role in regulating stress-responsive genes (Shinozaki et al., 2003). Here we show that the four expressed OsPR4 genes are significantly (3–9-fold) induced by the exogenous application of ABA (100 ␮M). It has been shown that ABA has negative roles in disease resistance (Mauch-Mani et al., 2005) and acts with an antagonistic effect on the JA-signaling pathway (Anderson et al., 2004; Mauch-Mani et al., 2005). ABA and biotic stress signaling do not always have opposing effects. The MYB-related protein BOTRYTIS SUSCEPTIBLE1 (BOS1) from Arabidopsis functions as a transcriptional activator in ABA signaling and is induced by Botrytis cinerea through the JA pathway (Mengiste et al., 2003). Similarly, CaCDPK3 is induced by ABA, SA, JA, ET, bacterial pathogen, and abiotic stresses (Chung et al., 2004). Our results indicate that the OsPR4 genes are induced both by ABA and JA, and ABA-responsive cis-elements are also found in the promoters of three OsPR4 genes. The expression levels of OsPR4 genes are also induced by different abiotic stresses including drought, salt, cold, heat shock, and UV light. However, the induction patterns of these genes to different stresses are extremely diverse, suggesting that the OsPR4 genes may have different contributions in response to these abiotic stresses. Together, these results suggest that the PR4 family might have a positive role in both abiotic and biotic stress responses, which may be mediated by ABA- and JA-signaling pathways. Although PR4 genes have been implicated in the stress response, no direct evidence has been reported for PR4 genes participating in drought tolerance in important crops such as rice. To confirm that the OsPR4 family is actually involved in drought tolerance, OsPR4a was overexpressed in rice, and the transgenic rice indeed showed better drought tolerance at the seedling and reproductive stages. We compared a few stress-related physiologic indices such as contents of superoxide dismutase and proline between overexpression lines and control plants after drought stress; however, no significant difference was found (data not shown). We also checked water loss rate (RWC of leaves) and changes of leaf water potential during the drought stress and no significant difference was found either (data not shown). These results suggest that novel mechanism may be involved in the improved drought resistance by OsPR4a-overexpression. Class II PR4 proteins have been shown to have RNase activity (Caporale et al., 2004; Bertini et al., 2009; Li et al., 2010). A report has shown some PR-10 proteins also have in vitro RNase activity that may act directly on the pathogens or protect plants during programmed cell death around infection sites (Park et al., 2004). At the same time, pea PR-10.1 can enhance the germination of Brassica napus under saline conditions (Srivastava et al., 2004) and WAP18, a PR-10 from mulberry, might function in the freezing–tolerance

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mechanism during the winter (Ukaji et al., 2004). It is possible that PR-10 protein might function as a LEA-like protein to protect plants from biotic and abiotic stresses (Liu and Ekramoddoullah, 2006). JIOsPR10 protein from rice possesses RNase activity, is induced by abiotic stresses, and might have a role in self-defense mechanisms against biotic and abiotic stresses in plants (Kim et al., 2008). Although many details for the mechanism of RNase in tolerance to abiotic stresses remain unclear, the facts that OsPR4 genes are responsive to abiotic stresses and OsPR4a overexpression lines showed increased drought tolerance in rice indicate that this family may play an important role in abiotic stress tolerance. Acknowledgments We thank Yanjun Kou for technical help on blast fungal inoculation. This work was supported by grants from the National Natural Science Foundation of China (30725021, 30830071, and 30921091), and the National Special Key Project of China on Transgenic research (2008ZX08001-003). References Agrawal GK, Jwa NS, Han KS, Agrawal VP, Rakwal R. Isolation of a novel rice PR4 type gene whose mRNA expression is modulated by blast pathogen attack and signaling components. Plant Physiol Biochem 2003;41:81–90. Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, Ehlert C, et al. Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell 2004;16:3460–79. Bertini L, Caporale C, Testa M, Proietti S, Caruso C. Structural basis of the antifungal activity of wheat PR4 proteins. FEBS Lett 2009;583:2865–71. Bertini L, Cascone A, Tucci M, D’Amore R, Di Berardino I, Buonocore V, et al. functional analysis of new members of the wheat PR4 gene family. Biol Chem 2006;387:1101–11. Bravo JM, Campo S, Murillo I, Coca M, San Segundo B. Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize. Plant Mol Biol 2003;52:745–59. Broekaert I, Lee HI, Kush A, Chua NH, Raikhel N. Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc Natl Acad Sci U S A 1990;87:7633–7. Caporale C, Di Berardino I, Leonardi L, Bertini L, Cascone A, Buonocore V, et al. Wheat pathogenesis-related proteins of class 4 have ribonuclease activity. FEBS Lett 2004;575:71–6. Caruso C, Caporale C, Poerio E, Facchiano A, Buonocore V. The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence. J Protein Chem 1993;12:379–86. Chen H, Wang S, Xing Y, Xu C, Hayes PM, Zhang Q. Comparative analyses of genomic locations and race specificities of loci for quantitative resistance to Pyricularia grisea in rice and barley. Proc Natl Acad Sci U S A 2003;100:2544–9. Chu ZH, Peng KM, Zhang LD, Zhou B, Wei J, Wang SP. Construction and characterization of a normalized whole-life-cycle cDNA library of rice. Chin Sci Bull 2003;48:229–35. Chung E, Park JM, Oh SK, Joung YH, Lee S, Choi D. Molecular and biochemical characterization of the Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) gene induced by abiotic and biotic stresses. Planta 2004;220:286–95. De Vos M, Van Oosten VR, Van Poecke RM, Van Pelt JA, Pozo MJ, Mueller MJ, et al. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol Plant Microbe Interact 2005;18:923–37. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, et al. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 2006;9:436–42. Guevara-Morato MA, de Lacoba MG, Garcia-Luque I, Serra MT. Characterization of a pathogenesis-related protein 4 (PR-4) induced in Capsicum chinense L3 plants with dual RNase and DNase activities. J Exp Bot 2010;61:3259–71. Jiang CJ, Shimono M, Sugano S, Kojima M, Yazawa K, Yoshida R, et al. Abscisic acid interacts antagonistically with salicylic acid signaling pathway in riceMagnaporthe grisea interaction. Mol Plant Microbe Interact 2010;23:791–8. Kim ST, Yu S, Kang YH, Kim SG, Kim JY, Kim SH, et al. The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity. Plant Cell Rep 2008;27:593–603. Li X, Xia B, Jiang Y, Wu Q, Wang C, He L, et al. A new pathogenesis-related protein, LrPR4, from Lycoris radiata, and its antifungal activity against Magnaporthe grisea. Mol Biol Rep 2010;37:995–1001. Lin YJ, Zhang Q. Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 2005;23:540–7. Linthorst HJ, Danhash N, Brederode FT, Van Kan JA, De Wit PJ, Bol JF. Tobacco and tomato PR proteins homologous to win and pro-hevein lack the hevein domain. Mol Plant Microbe Interact 1991;4:586–92.

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