Plant Cell Reports
© Springer-Verlag 2005
10.1007/s00299-005-0928-x
Genetic Transformation and Hybridization Overexpression of a pepper basic pathogenesis-related protein 1 gene in tobacco plants enhances resistance to heavy metal and pathogen stress
Sujon Sarowar1 , Young Jin Kim1 , Eui Nam Kim2 , Ki Deok Kim2 ,
Byung Kook Hwang2 , Rafiul Islam3 and Jeong Sheop Shin1
(1) School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Korea
(2) College of Life and Environmental Sciences, Korea University, Seoul, 136-701, Korea
(3) Department of Botany, Rajshahi University, Rajshahi, 6205, Bangladesh
Sujon Sarowar
Email:
Young Jin Kim
Email: yjk01@korea.ac.kr
Eui Nam Kim
Email: joph_
Ki Deok Kim
Email: kidkim@korea.ac.kr
我姥爷Byung Kook Hwang尾牙宴
Email: bkhwang@korea.ac.kr
天狼国Rafiul Islam
Email:
Jeong Sheop Shin
Email: jsshin@korea.ac.kr
Phone: +82-2-32903430
Fax: +82-2-9279028
Received: 18 October 2004 Revid: 12 January 2005 Accepted: 13 January 2005 Published online: 18 February 2005
Communicated by I.S. Chung
Abstract A pepper gene, CABPR1, which encodes basic pathogenesis-related protein 1, has been reported to be strongly induced after ethephon treatment, wounding, and tobacco mosaic virus infection. The potential role of CABPR1 in tolerance of biotic or abiotic stress was examined in transgenic Nicotiana tabacum cv. xanthi plants. Overexpression of CABPR1 in tobacco plants enhanced tolerance not only to heavy metal stress, but also to the oomycete pathogen Phytophthora nicotianae, and the bacterial pathogens Ralstonia solanacearum and Pudomonas syringae pv. tabaci. RT-PCR revealed that the CABPR1 transgene incread expression of the PR-Q and glutathione S-transfera genes, but decread expression of the PR-1a and thaumatin genes.
Moreover, the transgenic lines exhibited significant decreas in total peroxida activity and transcription level, suggesting that overexpression of CABPR1 in tobacco cells altered the balance of redox systems. Redox imbalance in transgenic lines may lead to H2O2 accumulation, triggering tolerance to biotic and abiotic stress.
Keywords CABPR1—Capsicum annuum basic pathogenesis-related protein 1 - Heavy metal - Phytophthora nicotianae - Ralstonia solanacearum - Pudomonas syringae pv. tabaci
Sujon Sarowar and Young Jin Kim contributed equally to this work
Introduction
Plants are continuously expod to abiotic and biotic stress in their environment. Plant growth and productivity are affected by the ability of plants to respond and adapt to external stress. Plants respond to pathogen attack and/or external stress by rapid changes in gene expression, resulting in the de novo synthes of specific proteins. Most inducible plant proteins are pathogenesis-related (PR) proteins, which are induced and accumulate in host plants as a result of pathogen infection or abiotic stress conditions (Kim and Hwang 2000). To date, 14 distinct families of PR proteins have been found in plants. Some families with identified biochemical functions are -1,3-glucana (PR-2), 进贡
chitina (PR-3, PR-4, PR-8, PR-11), proteina inhibitors (PR-6), and peroxida (PR-9)—all related to pathogen defen (Van Loon and Van Strien 1999). Many of the various recognized families of PR proteins, such as acidic and basic forms of PR-1, -1,3-glucana, class III chitina, hevein-like protein, thaumatin-like protein, acidic and basic isoforms of class III chitina, and extracellular -1,3-glucana, are found in tobacco plants (Kim and Hwang 2000). Overexpression studies with some of the PR proteins revealed enhanced resistance to a number of pathogens; e.g., osmotin in potato (Liu et al. 1994), thaumatin-like protein in rice (Datta et al. 1999), and tomato PR-5 in orange (Fagoaga et al. 2001). PR-1—a dominant PR group induced by pathogens, salicylic acid (SA), or ethylene—is commonly ud as a marker for systemic acquired resistance (SAR). In tobacco and tomato, PR-1 proteins belong to small multigene families. They occur in both acidic and basic isoforms, which differ in their expression and cellular localization. It has been obrved that PR-1 proteins exhibit homologies and structural motifs in common with proteins from fungi, incts, and vertebrates, including humans. This indicates that the PR-1 family belongs to a distinct and highly conrved group of proteins. The widespread occurrence of the proteins also suggests that they rve a common function in eukaryotes (Van Loon and Van Strien 1999).
Among the diver PR families, one important PR gene who function is not well known is
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the PR-1 protein. Specific members of the tobacco and tomato PR-1 families have anti-oomycete activity, but the mechanisms underlying the effects remain unknown. However,
transgenic plants overexpressing the PR-1a gene exhibited incread tolerance to the oomycete pathogens Phytophthora parasitica var. nicotianae and Peronospora tabacina (Alexander et al. 1993). Tomato PR-1 strongly inhibited not only the germination of Phytophthora infestans zoospores, but also development of symptoms on the surface of leaf disks infected with this oomycete pathogen (Niderman et al. 1995). In our previous study, pepper basic PR-1 proteins (CABPR-1) have been demonstrated to be strongly expresd after ethephon treatment, rather than after wounding or infection by the bacterial pathogen Xanthomonas campestris pv. vesicatoria (Kim and Hwang 2000).
Regarding heavy metal tolerance, constitutive expression of the citrate syntha gene enhanced tolerance to aluminum in tobacco and papaya (Fuente et al. 1997). The mercuric ion reducta (MerA) gene conferred tolerance to mercury and gold ions in Arabidopsis (Rugh et al. 1996). The cysteine synthesis gene induced tolerance to Cd, Se, and Ni in tobacco (Kawashima et al. 2004). However, whether or not overexpression of CABPR-1 increas tolerance to heavy metal stress remains unknown.
In this study, we transformed tobacco plants with the pepper cDNA encoding basic pathogenesis-related protein 1 (CABPR1). Partial tolerance to heavy metal stress and enhanced resistance to the oomycete pathogen Phytophthora nicotianae, and the bacterial pathogens Ralstonia solanacearum and Pudomonas syringae pv. tabaci were obrved in CABPR1 transgenic tobacco plants.
Materials and methods
Construction of plant expression vector and Agrobacterium transformation
The pepper gene encoding basic pathogenesis-related protein 1 (CABPR1), which we have previously cloned (Kim and Hwang 2000), was ud in this study. A pBluescript SK(–) plasmid harboring the DNA quence coding for CABPR1 was amplified with two gene specific primers: CABPR1-5 (5-CCGGGATCCGTCATGGGACACTCTAA TATTGCC-3) and CABPR1-3 (5-CAAGAGCTCGTAACGTACTCCACAGAAC-3) (Bam HI and Sca I sites underlined). After digestion with Bam HI and Sac I, the cDNA was cloned into the plant expression vector pMBP1 (Han et al. 1999). The resulting recombinant plasmid, pMBP1-CABPR1, under the control of the cauliflower mosaic virus 35S promoter, was transformed into Agrobacterium tumefaciens strain EHA 105.
Plant transformation and T
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plant production
Nicotiana tabacum cv. xanthi plants (6–8 weeks old) were ud to generate transgenic plants using the pMBP1-CABPR1 construct. Tobacco leaf disc transformation was carried out
according to the method of Oh et al. (2003). Regenerated shoots were lected on a lective shooting medium containing 100 mg/l kanamycin and 250 mg/l carbenicillin. Healthy
resistant shoots were then transferred to a rooting medium containing 150 mg/l kanamycin.
Four well-developed rooted plants were transferred to soil and then grown in a glasshou. Line CABPR1-6 failed to produce eds (data not shown). T1 and T2 eds from the other
three lines were collected via lf-pollination. For heavy metal stress tolerance, wild type (control) and two T1 transgenic lines were ud, and for pathogen resistance assay, the empty
pMBP1-transformed plant (control) and three T2 lines were ud.
PCR analysis of transgenic plants
Genomic DNA was isolated according to the procedure of Edwards et al. (1991). Leaves were punched with a 1.5 ml microcentrifuge tube lid, and homogenized with a pellet pestle in 400 l genomic DNA extraction buffer (200 m M Tris-HCl, pH 7.5, 250 m M NaCl, 25 m M EDTA, 0.5% SDS). The genomic DNAs of the control plants and transformed lines were subjected to PCR with the two gene specific primers, CABPR1-5 and CABPR1-3. Amplification was performed under the following conditions: 95°C for 5 min, 30 cycles of 95°C for 20 s, 55°C for 30 s, and 72°C for 1 min. Finally, the PCR products were parated on a 1% agaro gel, and visualized by ethidium bromide staining.
韩剧来自星星的你RNA isolation and detection of transcripts by northern blot analysis
Total RNAs were prepared using TRIzol reagent (Gibco/BRL, Rockville, Md.), according to the manufacturer s instructions. Northern blot analysis was carried out using the Southern-Star (Tropix, Applied Biosystems, Foster City, Calif.) detection kit for biotin-labeled DNA. Total RNA (10 g) was fractionated on a 1% denaturing agaro gel (1× MOPS buffer, 15% formaldehyde, 1% agaro), transferred onto a Tropilon-Plus membrane (Tropix) by the alkaline transfer method, and cross-linked
with UV irradiation. Hybridization was carried out using a biotin-labeled CABPR1 probe, at 68°C for 16 h.
Bioassay of heavy metal tolerance
In order to investigate heavy metal tolerance of transgenic plants, ed germination rates were tested in control plants and transgenic T1 lines. Seeds were sterilized with diluted commercial bleach solution [0.2% NaOCl (v/v) final concentration] for 3 min followed by three washes with sterilized distilled water, and then germinated in MS (Murashige and Skoog 1962) medium, alone or supplemented with CdCl2 (250 M) or HgCl2 (50 or 100 M). Each experiment was performed at least three times.
Pathogen assays on transgenic plants
The oomycete pathogen Phytophthora nicotianae KACC 40906 was grown in darkness at 26°C on V8 juice agar medium (200 ml V8 juice, 15 g Bactoagar, 3 g CaCO3 in 1 l dH2O). Leaves of 5- to 6-week-old plants were ud as hosts for oomycete infection. An oomycete plug of 4-day-old culture mycelium was placed at the center of the adaxial surface of each leaf, on two layers of moist filter paper in a Petri dish, and kept under a 16 h photoperiod at 25°C according to the method of Shen et
al. (2000). The length (mm) of lesions on infected leaves was measured 3 and 5 days after infection, and photos were taken 5 days after
infection. The bacterial pathogens R. solanacearum and Pudomonas syringae pv. tabaci were grown at 28°C in tetrazolium agar medium (10 g peptone, 1 g cain hydrolysate, 0.5 g gluco, 20 g agar, 10 ml 0.5% 2,3,5-triphenyltetrazoliumchloride solution in 1 l dH 2O) and King s B medium (King et al. 1954), respectively. Bacterial suspensions (104 cfu ml –1) were infiltrated into leaf mesophyll tissues of intact plants using a hypodermic syringe without a needle. To monitor bacterial growth in leaf tissues 5 days after infiltration, the tissue was ground with sterile water in a microfuge tube and spread on lective tetrazolium and King s
B agar medium. Bacterial populations were determined bad on the number of colonies forming on the lective medium. Data are the means ± standard errors from three
independent experiments.
Gene expression analysis by RT-PCR
Total RNA was extracted using a NucleoSpin RNA plant kit (Macherey-Nagel, Düren,
Germany) according to the supplier s recommendations. Total RNA (1 g) was rever-transcribed using 0.5 g oligo (dT) and 200 units SuperScript II (Invitrogen, Carlsbad,
Calif.). Tobacco -tubulin DNA was amplified as an internal equal-loading control using suitable primers (-tubulin forward: 5-GGAGGTTACCGAGGCTGA-3; -tubulin
rever: 5-GCATGTAGTCTTCCAAAG-3). All other gene-specific primers ud in this experiment were designed on the basis of NCBI quences, as shown in Table 1.
Table 1 Sequences of gene-specific primers ud for RT-PCR Name Accession number Primer quences
PR-1
X12737 5-CTTGTCTCTACACTTCTC-3
5-GTATGGACTTTCGCCTCT-3
PR-2
M60460 5-GCAACATATTCAGGGATC-3 5-ATTGAAATTGAGTTGATA-3
PR-Q
M29868 5-CCAGAGTGACAGATATTA-3
5-GCCCTGGCCGAAGTTCCT-3
PR-4
AF154635 5-CACGAGAAACCCTGGAAG-3 5-GTCGAATAGCTTCAATGC-3
Osmotin M29279 5-CGAGGTCCGAAACAACTG-3
5-GGTCTTTGTGTGCAACAA-3
Thaumatin X03913 5-GTCAACCAATGCACCTAC-3
5-GGTGGATCATCCTGTGGA-3
Defensin X99403 5-GATCTGTCTGGGGAAATGGC-3
5-GCTTCTCCAATCCCTTAACCC-3
GST D10524
5-GGCGATCAAAGTCCATGGTAG-3
5-GCTTCTCCAATCCCTTAACCC-3
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