Overexpression of a Pathogen-Inducible Myb Gene Increases Disease Resistance in Transgenic Rice 1

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1 BREEDING, GENETICS, AND PHYSIOLOGY Overexpression of a Pathogen-Inducible Myb Gene Increases Disease Resistance in Transgenic Rice 1 Min-Woo Lee and Yinong Yang ABSTRACT A jasmonic acid (JA)-inducible Myb transcription factor gene (JAmyb) was previously isolated from rice and shown to be closely associated with host cell death and fungal infection. To further elucidate the role of JAmyb in host cell death and defense response, the JAmyb cdna was fused to the constitutive 35S promoter and the stress-inducible rice PBZ1 promoter, respectively, and introduced into rice cultivar Drew by the Agrobacterium-mediated transformation. Inducible overexpression of JAmyb transgene led to host cell death and reddish lesions in leaves of transgenic rice. When infected with the rice blast fungus (Magnaporthe grisea), transgenic leaves with reddish lesions exhibited significantly enhanced blast resistance. Young transgenic seedlings, which had relatively low levels of JAmyb transgene expression but no reddish lesions, also showed increased tolerance to both rice blast and bacterial panicle blight (caused by Burkholderia glumae). These results suggest that JAmyb encodes a positive regulator of host cell death and may play a significant role in durable, broad-spectrum disease resistance. INTRODUCTION When plants recognize the invasion of pathogens during incompatible interactions, host defense mechanisms are generally triggered to inhibit the pathogen growth. Such processes often result in the hypersensitive response (HR), a form of rapid and 1 This is a completed study. 65

2 AAES Research Series 517 localized cell death that involves the activation of a complex array of defenserelated genes and anti-microbial compounds. Many studies have suggested that the cell death induced during the HR is likely a type of programmed cell death which is genetically programmed and essential for development and survival. This assumption is supported by evidence that the HR requires active plant metabolism, genetic control, and regulation of a defense-related biochemical pathway during resistant interaction. Therefore, there is a close connection between plant disease resistance and hypersensitive cell death. The Myeloblastosis viral oncogene (Myb) family of transcription factors in plants appears to have very diverse functions including regulation of phenylpropanoid, tryptophan biosynthesis, cellular morphogenesis, hormone-responsive pathways, and abiotic stress. Several plant myb genes were reported to be involved in cell death and defense response. A tobacco mosaic virus- and salicylic acid (SA)-inducible tobacco myb gene (myb1) was identified to be associated with viral infection and hypersensitive cell death (Yang and Klessig, 1996). At least two Arabidopsis myb genes were also shown to be induced by bacterial pathogens and associated with cell death (Kranz et al., 1998; Daniel et al., 1999). Overexpression of Atmyb30 in Arabidopsis and tobacco was shown to promote hypersensitive cell death and enhance disease resistance to fungal infection (Vailleau et al., 2002). We have previously isolated and characterized a blast- and JA-induced rice gene (JAmyb) that is closely associated with fungal infection and host cell death (Lee et al., 2001). In this study, we have generated transgenic rice plants with inducible overexpression of JAmyb transgene and examined its role in host cell death and disease resistance. PROCEDURES Gene constructs and rice transformation The overexpression construct was created by inserting the full cdna sequence into the vector pcambia1300 that contained either a double CaMV 35S promoter or a PBZ1 promoter. Transgenic constructs were introduced into Agrobacterium tumefaciens (strain EHA105) using a freeze-thaw method. The Agrobacterium-mediated transformation was performed using calli derived from mature embryos of the Drew cultivar (Oryza sativa spp. tropical japonica cv. Drew) according to the previously described method (Yang and Qi, 1999). Plant materials Hygromycin-resistant transgenic plants were grown to maturity in a 28 C greenhouse with 14/10 h (light/dark) cycle. Genomic southern analysis was conducted to determine the copy number of transgenic lines. After harvesting seeds from independent transgenic lines, transgenic plants that carry the transgene were selected by germinating seeds on filter paper soaked with 50µg/ml hygromycin. Positive selec- 66

3 B.R. Wells Rice Research Studies 2003 tion was confirmed by polymerase chain reaction (PCR) with primers corresponding to the PBZ1 promoter and 5 region of JAmyb using genomic DNA extracted from each transgenic plant. Homozygous transgenic lines identified from the second generation were used for further studies. Pathogen inoculation and disease assessment The fungal isolates used in this study belong to the IC-17 pathotype of M. grisea. The wild type IC-17-18/1 isolate (carrying avrpi-ta) is avirulent on Drew (carrying Pi-ta resistance gene) and its race-change mutant (IC-17-18/1-2, lacking avrpi-ta) is virulent on Drew (Harp and Correll, 1998). Two and one-half week-old control (nontransgenic) and transgenic plants were sprayed with a spore suspension (2x10 5 spores/ ml) of M. grisea containing 0.1% Tween 20. After being sprayed, plants were incubated in a dew chamber (22 C) for one day and moved to a growth chamber (28 C). Six days after blast inoculation, lesions were counted from 5-cm leaf tips of each plant and the three largest lesions were measured. Subsequently, the fungal growth was determined by hybridizing with a specific M. grisea rdna probe (Qi and Yang, 2002). For pathogenicity assays with Burkholderia glumae, about 20µl of bacterial suspension (10 7 cfu/ml) was injected using a needle into sheaths of 6 week-old transgenic plants. Lesion length and colony-forming units (cfu) were measured 7 days after bacterial inoculation. RNA extraction and gel blot analysis Total RNAs were isolated with TRIzol reagent (Invitrogen) from leaf tissues of control or transgenic plants after blast infection or chemical treatment. Ten µg of total RNA from each sample was separated on a 1.2% formaldehyde-agarose gel and transferred onto a nylon membrane and followed by UV-cross linking. The RNA gel blot was analyzed by hybridizing with the [α- 32 P] dctp-labeled gene-specific probe. Hybridization and washing were conducted in accordance with the PerfectHyb protocol (Sigma). RESULTS AND DISCUSSION Generation of transgenic rice with inducible overexpression of JAmyb transgene The JAmyb transgenic constructs were successfully introduced into commercial US rice cultivar Drew via the Agrobacterium-mediated transformation. Transformation efficiency (the number of regenerated plants/the number of calli cocultured with Agrobacterium x 100) was about 7% for the Drew cultivar. Transgenic calli carrying the 35S:JAmyb construct became dark brown and did not produce transgenic plants in the regeneration medium. In contrast, 27 primary transformants carrying the PBZ1:JAmyb construct that allowed inducible overexpression were successfully regenerated and 67

4 AAES Research Series 517 further characterized. The existence of the JAmyb transgene in the regenerated plants was identified by PCR and then confirmed by Southern analysis. Genomic Southern blots indicate that 23 transgenic lines carry one copy of the transgene whereas four lines contain two copies of the transgene. Inducible overexpression of JAmyb transgene leads to host cell death and lesion formation Endogenous JAmyb induction is closely associated with host cell death and lesion formation in rice plants; we therefore examined the effect of inducible overexpression of JAmyb transgene on host cell death. The chemical inducer benzothiadiazole (BTH, 250µM) was chosen and applied to leaves of 3 to 4 week-old transgenic plants because BTH can activate the JAmyb transgene controlled by the PBZ1 promoter but not the endogenous JAmyb gene. At 5 days after BTH treatment, large and chlorotic lesions were observed on the leaves of transgenic lines but not in the control plants. Relatively high expression of JAmyb transgene was also found in transgenic leaves with lesion formation. The rice PBZ1 promoter is not tightly controlled and is often induced by developmental or environmental changes in the greenhouse. As a result, one-third of the JAmyb transgenic lines developed reddish lesions at late vegetative stage or early reproductive stage (Fig. 1A). Gel blot analysis of RNAs from leaves of control and transgenic plants showed that expression of JAmyb transgene was increased especially in the lesion-containing regions (Fig. 1B). This lesion phenotype was inherited to the second (T1) and third (T2) generations. Homozygous T2 transgenic plants showed more extensive reddish lesions than that of heterozygous T2 plants. In addition, a higher level of JAmyb transgene expression was observed in the homozygous lines than in the heterozygous lines. We also found significant induction of phenylpropanoid biosynthetic genes (such as those encoding phenylalanine ammonia lyase, chalcone isomerase, flavanone hydroxylase, and flavonol synthase) and a higher level of kaempferol (a flavonol phytoalexin) in the JAmyb transgenic plants with reddish lesions. These results demonstrated that inducible overexpression of JAmyb transgene promoted host cell death and activated defense response in transgenic rice. Transgenic rice leaves with reddish lesions are highly resistant to blast infection To determine whether the transgenic rice with reddish lesions is resistant to blast infection, detached leaves were spot-inoculated with a virulent isolate of M. grisea. At 4 days after spot inoculation, extensive lesions with the fungal growth were observed in control plants. In contrast, large, necrotic lesions were not seen on leaves of the transgenic plants. Furthermore, the growth of M. grisea was about 4.5 times lower in transgenic lines than in control plants (Fig. 2). 68

5 B.R. Wells Rice Research Studies 2003 Transgenic rice seedlings exhibit enhanced tolerance to rice blast and bacterial panicle-blight pathogens To further evaluate the effect of JAmyb overexpression on disease resistance, homozygous T2 transgenic seedlings (two and one-half weeks old prior to lesion formation) were spray-inoculated with a virulent isolate of M. grisea. At 6 days after inoculation, transgenic seedlings had fewer lesion numbers than control plants (Fig. 3A). Lesions that developed in transgenic leaves also became more restricted and failed to enlarge while the rate of lesion expansion in the control was increased (Fig. 3B). In comparison with control plants, the growth of M. grisea was significantly reduced in transgenic lines (Fig. 3C), suggesting an increased level of blast resistance in JAmyb transgenic seedlings. When sheaths of control and transgenic rice plants were inoculated with a virulent strain of B. gluma, the lesion size in transgenic lines was significantly smaller than that of control plants (Fig. 4A). Transgenic rice lines also had much lower levels of bacterial growth than control plants (Fig. 4B). These data clearly demonstrated that JAmyb transgenic seedlings had increased tolerance to both fungal and bacterial infection. SIGNIFICANCE OF FINDINGS Rice blast and bacterial panicle blight are two of the most important rice pathogens in Arkansas and other southern rice-growing states. Arkansas rice cultivars are mainly dependent on major resistance genes such as Pi-ta for blast resistance. To date, no major genes have been identified for panicle blight resistance. Due to frequent race-change mutations (e.g., deletion of avrpi-ta) in the pathogen, new races may emerge to overcome Pi-ta and other major resistance genes, potentially resulting in a significant crop loss. Since JAmyb encodes a defense signaling component downstream of race-specific resistance genes, it is likely insensitive to race-change mutations and contributes to quantative, but more durable and broad-spectrum, resistance. Through the transgenic approach, our study demonstrates that genetic manipulation of pathogen-inducible transcription factors may provide a novel strategy for controlling rice diseases. ACKNOWLEDGMENTS This work is supported by the Arkansas Rice Research and Promotion Board. LITERATURE CITED Daniel, X., C. Lacomme, J.-B. Morel, and D. Roby A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death. Plant J. 20:

6 AAES Research Series 517 Harp, T.L. and J.C. Correll Recovery and characterization of spontaneous, selenate-resistant mutants of Magnaporthe grisea, the rice blast pathogen. Mycologia 90: Kranz, H.D., m. Denekamp, R. Greco, H. Jin, A. Leyva, R.C. Meissner, K. Petroni, A. Urzainqui, M. Bevan, C. Martin, S. Smeekens, C. Toneli, J. Paz-Ares, and B. Weisshaar Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 16(2): Lee, M.W., M. Qi, and Y. Yang A novel jasmonic acid-inducible rice myb gene associates with fungal infection and host cell death. Mol. Plant-Microbe Interact. 4: Qi, M. and Y. Yang Quantification of Magnaporthe grisea during infection of rice plants using real-time PCR and northern blot/phosphoimaging analysis. Phytopathology 92: Vailleau, F.A., X. Daniel, M. Tronchet, J.-L. Montillet, C. Triantaphylides, and D. Roby R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc. Natl. Acad. Sci. U S A. 99: Yang, Y. and D.F. Klessig Isolation of characterization of a TMV-inducible myb oncogene homologue from tobacco. Proc. Natl. Acad. Sci. USA 93: Yang, Y. and M. Qi Production of transgenic rice plants using Agrobacterium-mediated transformation. In: R.J. Norman and C.A. Beyrouty (eds.). B.R. Wells Rice Research Studies University of Arkansas Agricultural Experiment Station Research Series 476: Fayetteville, Ark. 70

7 B.R. Wells Rice Research Studies 2003 A CK H e te ro -T Hom o-t He-T (-) He-T (+ ) CK Ho-T (-) Ho-T (+ ) B JA m yb rrna Fig. 1. Lesion formation and JAmyb expression in transgenic plants. A. Spontaneous lesion formation in the JAmyb transgenic plants. Note the more extensive reddish lesions in homozygous lines (Homo-T) than heterozygous (Hetero-T) lines. B. Inducible overexpression of JAmyb transgene in leaf segments with reddish lesions. Note homozygous lines (Ho-T) show a higher level of JAmyb expression than heterozygous lines (He-T). CK: control, (-): No lesion, (+): Lesion. 100 Relative Fungal Growth CK T Fig. 2. Inhibition of M. grisea growth in JAmyb transgenic leaves with reddish lesions. Detached leaves of two-month-old control (CK; without lesion) and transgenic plants (T; with reddish lesions) were spot-inoculated with a virulent isolate of M. grisea. Levels of the fungal growth were determined at 6 days after spot inoculation. 71

8 AAES Research Series 517 Fig. 3. Enhanced resistance of JAmyb transgenic seedlings to M. grisea. A. Comparison of lesion number in control (CK) and transgenic (T11, T12, and T13) lines. B. Comparison of lesion length in control and transgenic lines. C. Relative growth of M. grisea in control and transgenic lines. CK: control. T11, T12, T13: three transgenic lines. Lesion Length A Log 10 (C F U /5 c m ) 10 B CK T11 T12 T13 CK T11 T12 T13 Fig. 4. Increased resistance of JAmyb transgenic plants to B. glumae. A. Lesion length on the sheath of control (CK) and three transgenic (T11, T12, and T13) lines. B. Bacterial growth in the sheath of control and three transgenic lines. 72