World J Microbiol Biotechnol DOI 10.1007/s11274-013-1546-3

ORIGINAL PAPER

Co-expression of RCH10 and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia solani and blast Magnorpathe oryzae and reveals impact on seed germination Bizeng Mao • Xuehui Liu • Dongwei Hu Debao Li

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Received: 29 July 2013 / Accepted: 29 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Rice sheath blight and blast caused by Rhizoctonia solani Ku¨hn and Magnorpathe oryzae respectively, are the two most destructive fungal diseases in rice. With no genetic natural traits conferring resistance to sheath blight, transgenic manipulation provides an obvious approach. In this study, the rice basic chitinase gene (RCH10) and the alfalfa b-1,3-glucanase gene (AGLU1) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate a double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation. More than 160 independent transformants were obtained and confirmed by PCR. Northern analysis of inheritable progenies revealed similar levels of both RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was challenged in open field inoculation. Immunogold detection revealed that RCH10 and AGLU1 proteins were initially located mainly in the chloroplasts and were delivered to the vacuole and cell wall upon infection, suggesting that these subcellular compartments act as the gathering and execution site for these anti-fungal proteins. We also observed that transgenic seeds display lower germination rate and seedling vigor, indicating that defense enhancement might be achieved at the expense of development.

B. Mao (&)  X. Liu  D. Hu  D. Li State Key Laboratory of Rice Biology and Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China e-mail: [email protected]

Keywords Transgenic rice  Rice basic chitinase gene  Alfalfa b-1,3-Glucanase gene  Sheath blight  Blast  Immunogold detection

Introduction Rice (Oryza sativa L.) is one of the most important food crops, providing staple diet for more than one half of the world population especially in developing countries. Rice production is often challenged due to several destructive diseases. Among them, sheath blight caused by Rhizoctonia solani Ku¨hn (R. solani), a soil-borne fungal pathogen with a broad host range without effective genetic resistance, causes severe yield loss from 8 to 50 % (Savary et al. 2000). Rice blast caused by Magnaporthe oryzae occurs worldwide, infects plants at all growth stages and tissues, also leads to severe yield loss (Talbot 2003). Although fungicides are usually used in the control of these fungal diseases, concerns over human health and environment pollutions demand for alternative strategies to enhance crops resistance against invading pathogens. Plant genetic engineering has been practiced as a promising strategy to provide resistance against diseases that pose big threats to economically important crops. During infection, plants produce a number of pathogenesis-related (PR) proteins to restrict pathogenic growth, many of these proteins have been found to be chitinases and b-1,3-glucanases (Broglie et al. 1986; Zhu and Lamb 1991; Shah and Klessig 1996; Gijzen et al. 2001; Thimmapuram et al. 2001; Park et al. 2004; Hoster et al. 2005; Nakazaki et al. 2006). Chitinase (EC 3.2.1.14) catalyzes the hydrolysis of b-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin which is absent in higher plants (Zhu and Lamb 1991). b-1,3-glucanases (EC

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3.2.1.39) are abundant hydrolytic enzymes widely distributed in plant, they hydrolyze b-1,3-linked glucans, major component of the cell walls of fungal pathogens, and act synergistically with chitinase to inhibit fungal growth (Maher et al. 1993). The degraded products in turn act as elicitors that are involved in inducible defenses of plants. In addition, polymers of 1,3:1,6-b-glucans are found only in fungi, but polymers of 1,3-b-glucans are found in both fungi and plants. Although our interest lies in the defensive actions of these enzymes, recent reports have linked chitinase and b-1,3-glucanases to diverse physiological and developmental processes such as microsporogenesis (Bucciaglia and Smith 1994; Dong and Dunstan 1997; Delp and Palva 1999) and seed germination (Leubner-Metzger and Meins 2000). De Jong et al. (1992) described the involvement of chitinase in embryogenesis. The availability of PR genes and their anti-fungal functions have led to their deployment in increasing disease resistance in crops. Previous studies have demonstrated that chitinase or/and glucanase transgenic plants exhibit enhanced resistance. Successful crops include rice (Lin et al. 1995; Datta et al. 2000; Maruthasalam et al. 2007), wheat (Anand et al. 2003), tobacco (Broglie et al. 1991; Neuhaus et al. 1992; Chye et al. 2005), strawberry (Asao et al. 1997) and oilseeds (Grison et al. 1996). Since disease resistance is a complex multigenic trait, a single transgene, in most case, only increases resistance marginally or enhances resistance in a non-specific manner. Chitinase and glucanase have been shown to exert antifungal activity synergistically in vitro (Mauch et al. 1988). Hybrid plant of transgenic tobacco expressing both b-1,3glucanase and chitinase genes produced enhanced resistance to fungal pathogens, compared to plants expressing only b-1,3-glucanase or chitinase gene singly (Zhu et al. 1994). These findings indicate that the combined expression of chitinase and b-1,3-glucanase provides effective protection against fungal pathogens. Our preliminary study has also shown that the combined expression of RCH10 and AGLU1 could enhance resistance to sheath blight in transgenic rice under controlled greenhouse conditions (Mao et al. 2003). Significant advances in gene delivery techniques have allowed the incorporation of genes conferring specific agronomic traits into many crop plants (Dale et al. 1993). While genetic engineering of rice has made marked advances in the past few years, there are cases with undesirable effects such as transgenic Taipei 309 with nptII and IR72 and Koshihikari with bar yielded poor field performance, limiting their potential commercial value (Tu et al. 2000). Oard et al. (2000) reported that glufosinate herbicide resistance transgenic lines suffered significant loss in plant height and maturity compared to non-transgenic material.

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Our previous work has shown that the transgenic rice (cv. Taipei 309) co-transformed with RCH10 and AGLU1 exhibits enhanced resistance to rice sheath blight under greenhouse condition. In this study, upon obtaining sufficient transgenic plants (cv. Taipei 309) expressing RCH10 and AGLU1, we conducted further investigations on these transgenic lines in field trials, and showed that combined transgenes expression is heritable in some lines within generations. We observed that transgenic seeds displayed lower germination rate and seedling vigor, and expression of transgenes gave rise to enhanced resistance to both sheath blight and blast. Interestingly, we found that the RCH10 and AGLU1 proteins appeared to accumulate in chloroplasts and migrated to the vacuole and cell wall upon pathogen infection, revealing a possible route of anti-fungal mechanism.

Materials and methods Plasmid construction and plant transformation Previously, the rice basic chitinase gene (RCH10) (Zhu and Lamb 1991) and alfalfa b-1,3-glucanase gene (AGLU1) (Maher et al. 1993) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate double-defense gene expression cassette pZ100 (Zhu et al. 1994, provided by Dr. Qun Zhu, the Salk Institute for Biological Sciences, CA). The construct was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation of embryogenic callus derived from mature embryos generating more than 160 independent transformants. T0 plants were transferred to greenhouse containment conditions and selected on the basis of transgene expression and integration. Homozygous transgenic lines in the T2 generation were identified and stable lines were obtained in the fifth generation (T5). All plants were grown at 27 ± 1 °C under 18 h/6 h light/dark photoperiod. RNA preparation and northern blot Total RNA was isolated from the leaves of transgenic and wild type (WT) plants using Trizol reagent according to the manufacturer’s protocol (GIBCO BRL Life Technologies, Gaithersburg, MD). Twenty microgram of RNA was fractionated on a 1 % formaldehyde-denaturing agarose gel and blotted onto Hybond-NC membranes (Amersham Piscataway, NJ), in which was hybridized with randomly labeled probes RCH10, as described (Ning et al. 2004); AGLU1 was amplified from pZ100 by PCR using the following primers: 50 -GGT GTG CCT AAT TCC GAC CTT C-30 and 50 -GTA GCC CAA GGC CTT CTT GGA

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G-30 . Hybridization signals were detected by phosphor imaging using a Typhoon 9200 imager (Amersham Pharmacia). In vivo bioassays To test resistance against R. solani transgenic plants were infected with R. solani race AG-1-1A as described by Pan et al. (1997). Briefly, 5 to 6-day old seedlings were transferred to nutrient solutions and grown to maximum tillering stage to be used for inoculation. The rice sheath blight pathogen R. solani was maintained on a potato-dextrose agar (PDA) plate as described by Pan et al. (1999). On the following day, a 5-mm agar disk from the periphery of an actively growing colony of R. solani, held in place by sterile toothpicks, 1 cm in length, was transferred to a fresh PDA plate. The plates were incubated at 28 °C until the toothpicks were colonized by the pathogen. Plants were inoculated at tillering stage by placing the 1-cm R. solani colonized toothpick into the lowest inner sheath of the main tiller. The plant infected with R. solani was also used as donor to test resistance to M. grisea. Another tiller was inject-inoculated with M. grisea race ZB15 as described previously (He et al. 1992). After inoculation, the plants were immediately transferred to growth chamber and kept at 27 ± 1 °C with 90 % relative humidity. A total of 15 plants were sampled for each independent transformant. Sheath blight symptom was measured from 5 days post inoculation (dpi) up to 14 dpi by measuring the length of lesions. Blast disease was scored from 3 to 7 dpi by measuring lesions dispersion. All experiments were conducted under greenhouse conditions. The independent field trials were conducted near the Agricultural Farm of Zhejiang University in Zhejiang District, Hangzhou, the People’s Republic of China. To test resistance, the T4 and T5 plants were infected with R. solani and M. grisea until tillering stage, the inoculation methods and evaluations of resistance as described above. These experiments were carried out at least three times with the independent lines harbouring RCH10 and AGLU1 genes, to confirm the reproducibility of the results. Enzyme activity assay Leaf tissues from transgenic plants and control plants were collected after induction with R. solani at different times, immediately weighed and frozen in liquid nitrogen and stored at -20 °C. Crude protein was extracted according to the method of Mao et al. (2012). Typically, 200 mg leaves devoid of main midrib were homogenized with 2 % PVPP and 50 mM HEPES extraction buffer (pH 7.8) containing 0.2 mM EDTA and 2 mM ascorbic acid with a pre-chilled motor and pestle. The homogenate was then centrifuged at

12,000g for 20 min at 4 °C. The supernatant was used as crude extract for enzymatic assays. All protein concentrations of the crude extract were determined according to Bradford (1976). Chitinase (EC 3.2.1.14) was assayed by monitoring the release of N-acetyl-D-glucosamine (NAGA), Reactions were carried out at 37 °C. One unit of specific activity was defined as micromoles of NAGA produced per minute per milligram of protein. b-1,3-glucanase (EC 3.2.1.39) was assayed by monitoring the release of free glucose from laminarin with glucose oxidase reagent (Sigma) according to the manufacturer’s directions. Reactions were carried out at 37 °C. One unit of specific activity was defined as micromoles of glucose released per minute per milligram of protein. Each assay was repeated four times, the experiment was repeated three times, and data obtained were pooled and averaged. Immunogold detection Young leaves were put on the filter papers soaked with 50 mM phosphate buffer (pH 7.2) and inoculated with the agar disc (diameter 3 mm) covered with sheath blight hyphae. Inoculated leaves were kept at 28 °C. Leaves were fixed in 50 mM phosphate buffered saline (PBS; pH 7.2) containing 1 % (v/v) glutaraldehyde and 2 % (v/v) polyformaldehyde overnight at 4 °C. Samples were washed three times with PBS and post-fixed with 1 % (w/v) osmium tetroxide in PBS for 2 h at room temperature. After three rinses in MilliQ water the samples were dehydrated in a graded ethanol series, 30 % at 4 °C, and 50, 70, 90, 100, 100 % at 28 °C, respectively, and embedded in Lowicryl K4M resin (Electron Microscopy Sciences, Fort Washington, PA). Samples were polymerized slowly under UV irradiation for 3 days at -20 °C before placement at room temperature for another 3 days. Sections (70 nm) of leaves were cut with a diamond knife and mounted on nickel grids. After blocking for 30 min at room temperature with 50 mM PBS (pH 6.8) containing 1.0 % bovine serum albumin and 0.02 % polyethylene glycol 2000 (blocking solution), the grids were incubated for 1 h in polyclonal antibody raised against the RCH10 and AGLU1 proteins for 20–60 min at room temperature, respectively. The grids were washed twice in PBS and three times in blocking buffer before a 1 h incubation with Protein A-gold (Sigma, St. Louis, MO) diluted in blocking solution. After incubation, the grids were washed twice in PBS and three times in MilliQ water before staining with uranyl acetate and lead citrate. The sections were viewed by electron microscopy (JEM-1200EX; JEOL, Japan). Control experiment included replacing polyclonal antibodies against the RCH10 and AGLU1 protein with preimmune serum prior to incubation with Protein A-gold. A

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Fig. 1 Schematic representation of the transformation vector pZ100. pZ100, the double-defense genes expression cassette, derived from plant transformation vector pBI101 tandemly inserted with CaMV 35S enhancer/RCH10/NOS and a double CaMV 35S promoter/ AGLU1/NOS fusion fragment. RB right-border, LB left-border, 35S

CaMV 35S promoter, 35S En CaMV 35S enhancer, NOS terminator of the nopaline synthase gene, NPT II coding region of the neomycin phosphor-transferase gene, AGLU1 coding region of the AGLU1 gene, RCH 10 coding region of the RCH 10 gene

total of 5 plants were sampled for each treatment and time interval.

plants followed the expected Mendelian segregation pattern of 3:1 (data not shown). Double resistance of the T1 plants was evaluated using a strong virulent R. solani strain AG-1-1A and M. grisea race ZB15. As expected, most of the transgenic lines exhibited delayed and reduced disease symptoms compared with those of WT Taipei 309 (Fig. 2a–d). Homozygous transgenic lines in the T2 generation were identified and stable resistance lines were obtained in the next generation, T4 and T5 plants were infected with R. solani and M. grisea under open-field condition, the leision observed in T4 and T5 plants (transgenic lines J6 and J161) were similar to those of T1 (Fig. 2e, f). These results indicated that expression of the RCH10 and AGLU1 genes in most transgenic plants conferred enhanced resistance to both rice sheath blight and blast fungus. Expression of RCH10 and AGLU1 in the transformants was investigated by northern blotting. Expression levels varied among the transformants (Fig. 3a). Based on both gene expression and diseases resistance, four lines (lines 6, 61, 63 and 121) were selected to trace the hereditability of transgene expression and disease resistance in following generations. In T5, two stable transgenic lines (J6, J61) were obtained (Fig. 3b).

Seed germination and seedling growth conditions Mature dry seeds were incubated on filter paper wet with sterilized water in 9-cm Petri dishes. The dishes were sealed to prevent evaporation and incubated in a controlled temperature chamber in darkness at 25 °C. Sprouted seeds were immediately transferred to a green house. Phenotypic characterizations were scored by measuring plant height. A total of 100 seeds were sampled for each independent transformants, the experiment was repeated three times. Statistical analysis All treatments were conducted in a randomized complete block design. For each enzymatic analysis, 5 independent tissue samples were used. Data was expressed as mean ± standard deviation (SD). Duncan’s multiple-range test (SSR) was used with the Data Processing System (DPS) Statistical Software package (Tang and Feng 2002).

Results Generation of transgenic lines with inheritable disease resistance to both sheath blight and blast The expression cassette pZ100 contains double cauliflower mosaic virus (CaMV) 35S promoter placed upstream of the 1,387 bp full length AGLU1 cDNA sequence, the nopaline synthase (NOS) terminator and the enhancer region of the CaMV35S promoter positioned upstream of the 1, 400 bp promoter and coding region of RCH 10 and the NOS terminator tandemly inserted into the plant transformation vector pBI101 (Fig. 1). Transgenic rice (Oryza sativa L. cv. TP309) was produced by Agrobacterium tumefaciens-mediated transformation, using the geneticin (G418) resistance gene as selectable marker. A total of 166 independent transgenic lines were shown to have the double transgenes. Both G418 resistance and transgenes sequences in selfed progeny (T1) transgenic plants demonstrated that many of the progeny

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Effect of transgenes on chitinase and b-1,3-glucanase activity Transgenic lines J6, J61 and WT plants were assayed for Chitinase and b-1,3-glucanase activities. Leaf tissues from transgenic and WT plants were harvested at 0, 24, 48, 96, 120 h after induction with R. solani. Effect of transgenes on Chitinase and b-1,3-glucanase activities were shown in Fig. 4. During the 120 h time course, both transgenic lines exhibited significantly higher chitinase activity than WT (P \ 0.01) while each sample maintained little change during inoculation time. In addition, a highly significant difference (P \ 0.01) was detected between J6 and J61. At 0 post-infection, the Chitinase activities of J6 and J61 were approximately 16-fold and 21-fold greater than that in WT, respectively. The change of b-1,3-glucanase activity was similar to that observed in Chitinase. These results implied that transgenes enhanced Chitinase and b-1,3-glucanase activity.

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Fig. 2 Resistance in transgenic rice plants that constitutively overexpressed AGLU1 and RCH 10 gene. a Lesion lengths of independent transgenic lines (T1) inoculated with R. solani AG-1-1A and wild-type 309 as control. Lesion length was measured at 3 days (white column) and 14 days (pattern column) post inoculation (dpi). Error bars indicated standard deviations from three independent experiments. b Typical sheath symptoms observed on 14 days post inoculation (dpi) with R. solani AG-1-1A on transgenic (T1) and wild-type 309 (WT) plants. c Lesion lengths of independent transgenic lines (T1) inoculated with M. grisea ZB15 and wildtype 309 as control. Lesion sizes were measured at 3 days (white column) and 7 days (pattern column) post inoculation (dpi). Error bars indicated standard deviations from three independent experiments. d Typical leave symptoms observed at 7 days post inoculated (dpi) with M. grisea ZB15 on transgenic (T1) and wild-type 309 (WT) plants. e Typical sheath symptoms were observed with R. solani AG-1-1A on transgenic (T5) and wild-type 309 (WT) plants. f Typical leave symptoms were observed with M. grisea ZB15 on transgenic (T5) and wild-type 309 (WT) plants. WT, wildtype; J2, J6, J18, J31, J33, J61, J63, J76, J121 and J161, independent transgenic lines

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World J Microbiol Biotechnol Fig. 3 Northern blot analysis of transgenic lines expressing AGLU1 and RCH 10 gene. a AGLU1 and RCH 10 expression in T1 plants. b AGLU1 and RCH 10 expression in T5 homozygous progenies

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Subcellular localization and relocation of RCH10 and ALGU1 proteins To determine the subcellular localization of the RCH10 and AGLU1 proteins, we performed immunogold detection using polyclonal antibodies raised against the RCH10 or AGLU1. RCH10 protein was observed to be mainly accumulated in the chloroplasts, with weak signals detecting endogenous RCH10 in the chloroplasts of WT cells (Fig. 5a, b). Interestingly, immuno-signal of AGLU1 was also observed in the WT chloroplasts (Fig. 5f), suggesting that the AGLU1 antibody could detect the rice endogenous glucanase(s). Confirming the specificity of immno-signals that we obtained, control experiment with preimmune antiserum did not detect any gold particles in the sections (data not shown). Therefore, the RCH10 and AGLU1 proteinsreside in the chloroplasts. It is intriguing

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that both RCH10 and AGLU1 appeared to be translocated to the cell wall and vacuole after pathogen infection (Fig. 5c, d, g, h). This probably suggests that the relocation of anti-fungal proteins might denote important biological significance: there proteins are required at the sites when fungus invades and grows. Transgene inhibits seed germination and seedling growth Although mature transgenic plants did not exhibit unusual morphological changes and produced normal seed setting (data not shown), we observed that seed germination of the transgenic lines was significantly inhibited across all generations we followed (Fig. 6a). After germinating, the growth of transgenic seedlings were also delayed in comparison with WT (Fig. 6b–d). Growth of the transgenic

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Fig. 5 Immunoelectron microscopy with polyclonal antibodies against RCH10 and AGLU1. a Wild type leave hybridized with RCH10 polyclonal antibody. b Transformed J6 line hybridized with RCH10 polyclonal antibody. c, d The leaf of transformed J6 line 1 day post-infection with pathogen hybridized with RCH10

polyclonal antibody. e Wild type leaf hybridized with AGLU1 polyclonal antibody. f The leaf of transformed J6 line hybridized with AGLU1 polyclonal antibody. g, h The leaf of transformed J6 line 1 day post-infection with pathogen hybridized with AGLU1 polyclonal antibody. CW cell wall, V vacuole, and Ch chloroplast

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Fig. 6 Seed germination and phenotypic investigations of transgenic plants expressing RCH 10 and AGLU1 genes. a–e Growth of wild-type and transgenic lines J6 and J61 after germination 2, 4, 8, 16 and 18 days, respectively

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seedlings only caught up with those of WT plants after 18 days (Fig. 6e).

Discussion Resistance to rice sheath blight is usually inherited as a quantitative trait (Li et al. 1995; Zou et al. 2000; Sato et al. 2004; Pinson et al. 2005; Sharma et al. 2009; Channamallikarjuna et al. 2010). Although a large number of rice cultivars has been screened for resistance to R. solani, only few candidate resistant cultivars have been isolated, with no highly resistant rice germplasm being identified to be used in rice breeding to date (Pinson et al. 2005). For example, Jasmine 85 confers moderate resistance to some isolates of R. solani and several QTLs for resistance have been mapped in the variety (Pan et al. 1999; Venu et al. 2007). Resistance complexity and the lack of reliable inoculation method lead to no direct evidence to support the underlying resistant mechanisms in defense against sheath blight. On the other hand, blast resistance in rice has been extensively studied. To our knowledge, more than 50 major blast R genes have been reported (Chen et al. 2005; Liu et al. 2007). However, because of a high pathogenic diversity, resistance to blast is not durable in cultivated rice varieties. The aim of the current study is to examine the possibility and effectiveness of introducing durable disease resistance through gene manipulation in rice. Previous studies have indicated that transgenic coexpression of more than one defense genes could synergistically confer enhanced protection over the expression of single defense proteins (Zhu et al. 1994; Jongedijk et al. 1995; Anand et al. 2003; Kalpana et al. 2006). These findings prompted us to investigate whether the coexpression of RCH10 and AGLU1 in transgenic rice could enhance the levels of resistance to fungal diseases in open field environment. The major obstacle of the study was to obtain enough lines with stable inheritance and expression of the transgenes. Our transgenic lines revealed variable and contradictory results with regards to disease resistance. Among the 166 independent T0 transgenic lines identified, only 38 lines showed stable inheritance of the transgenes and significantly increased resistance to both R. solani and M. grisea resistance in the T5 progenies. The other 128 lines either did not express the transgene or expressed low levels of RCH10 and AGLU1 that did not ensure an increased resistance due to dose-dependent resistance feature. It has long been shown that disease resistance is often associated with certain developmental processes (Lynch et al. 1995; Oard et al. 1996, 2000; Tu et al. 2000). In Arabidopsis, it was recently well documented that the auxin pathway in development is strongly regulated during

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pathogen attack (Thilmony et al. 2006). The Arabidopsis microRNA miR393a represses auxin signaling through down-regulating the auxin receptor genes, resulting in increased resistance to P. syringae (Navarro et al. 2006). Furthermore, auxin production is proven to be a susceptible factor during pathogen challenge (Zhang et al. 2007). Our present study indicates that disease resistance is also closely associated with development in rice. Further understandings of the mechanism involved in the cross talk between disease resistance and other agronomic traits will provide a practical approach to manipulate disease resistance with other desirable agronomic characters. The subcellular location of RCH10 and AGLU1 were detected by immunogold technique, which intriguingly showed that both proteins were normally accumulated in the chloroplasts instead of the vacuole, a more commonly believed organelle (Zhu and Lamb 1991). We found that they were only relocated to the vacuole and cell wall after infection (Fig. 5). Our findings probably suggest a novel mechanism for defense protein function during pathogen attack. We propose that following pathogen invasion, defense proteins were somehow triggered and moved to the assaulted sites where anti-fungal action is demanded. However, the biological importance of their accumulation in the chloroplasts of healthy cells remains unclear. Nevertheless, the present study demonstrates a successful introduction of double defense genes into rice, resulting in enhanced resistance to agronomical important fungal pathogens. The transgenic lines obtained in this study expand the rice germplasm pool for rice breeding, achieving better disease management. Further investigation of the defense protein relocation and function and regulation of transcriptome may provide new insights into the molecular mechanisms involved in rice defense responses. Acknowledgments We appreciate Dr. Zuhua He for providing help in the study. We thank LO LI JANE for proofreading the manuscript. This work was partially funded by the Natural Science Foundation of Zhejiang Province (No. Y306253), the National Natural Science Foundation of China (Grant No. 90817102) and National Special Foundation for Transgenic Species of China (2011ZX08009-003-001, 2013ZX08009003-001).

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