Planta (2005) 221: 386–393 DOI 10.1007/s00425-004-1459-3
O R I GI N A L A R T IC L E
Yuan-Li Chan Æ Venkatesh Prasad Æ Sanjaya Kuei Hung Chen Æ Po Chang Liu Æ Ming-Tsair Chan Chiu-Ping Cheng
Transgenic tomato plants expressing an Arabidopsis thionin (Thi2.1) driven by fruit-inactive promoter battle against phytopathogenic attack Received: 26 October 2004 / Accepted: 22 November 2004 / Published online: 19 January 2005 Springer-Verlag 2005
Abstract Tomato is one of the most important crop plants; however, attacks by pathogens can cause serious losses in production. In this report, we explore the potential of using the Arabidopsis thionin (Thi2.1) gene to genetically engineer enhanced resistance to multiple diseases in tomato. Potential thionin toxicity in fruits was negated by the use of a fruit-inactive promoter to drive the Thi2.1 gene. In transgenic lines containing RB7/Thi2.1, constitutive Thi2.1 expression was detected in roots and incidentally in leaves, but not in fruits. Disease assays revealed that the transgenic lines that were tested conferred significant levels of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW). Further studies indicated that BW disease progression in transgenic lines was delayed by a systemic suppression of bacterial multiplication. By adopting a safe genetic engineering strategy, the present investigation is another step forward demonstrating thionin practicality in crop protection. Keywords Arabidopsis thionin Æ Disease resistance Æ Fruit-inactive expression Æ Tomato
Introduction In nature, plants constantly encounter a wide range of microbes. Agricultural productivity has always been at the mercy of pathogens, with losses surging to ecoY.-L. Chan Æ V. Prasad Æ Sanjaya Æ M.-T. Chan (&) C.-P. Cheng Institute of BioAgricultural Sciences, Academia Sinica, Taipei, 115, Taiwan E-mail:
[email protected] Tel.: +886-2-26516194 Fax: +886-2-26511164 E-mail:
[email protected] Tel.: +886-2-26522268 Fax: +886-2-26515600 K. H. Chen Æ P. C. Liu Eexon Science Inc., Gui Shan Country, Taoyuan, 333, Taiwan
nomically damaging levels during an outbreak. Plants constantly battle against pathogenic attacks through reinforcement of their complex defense networks, comprised of physical and biochemical barricades that govern active and passive modes of resistance. In addition, pesticide usage has been employed as an efficient approach for crop disease control. However, increasingly pathogens are becoming resistant to endogenous genes and pesticides (Rommens and Kishore 2000). Development of effective disease resistance to a broad range of pathogens in crops is certainly of importance, but usually requires tremendous resources and efforts when traditional breeding approaches by crossing are taken. In recent years, genetic engineering of disease resistance into crops has become valuable in terms of cost saving, efficacy, and reduction of pesticide usage. Particularly, the use of many potent anti-microbial peptides from plant resources for crop protection, such as enzyme inhibitors, lectins, pathogenesis-related proteins, and thionins, has been demonstrated by transgenic approaches (Bohlmann 1994). Thionins, 5 kDa, basic, cysteine-rich anti-microbial peptides, have been identified in a large number of plant species, either expressed constitutively or induced by pathogen attack or elicitors (Epple et al. 1997, 1998; Fernandez de Caleya et al. 1972). Thionins are active against a broad spectrum of phytopathogens, as demonstrated by toxicity studies and transgenic approaches (Bohlmann et al. 1988; Carmona et al. 1993; Epple et al. 1997; Holtorf et al. 1998; Molina et al. 1993; Terras et al. 1995). However, their use has been confined to homologous or model systems, not extended to crop improvement. This may be attributable to the fact that thionins, in general, are considered toxic to animals, as demonstrated in laboratory animals and mammalian cells in vitro (Bohlmann 1994; Bussing et al. 1999). This prompted us to investigate the use of thionins to genetically engineer disease resistance in crops, by adopting a safe and careful engineering strategy. Tomato (Lycopersicon esculentum) is a natural host to a broad spectrum of pathogens, including Fusarium
387
oxysporum f. sp. lycopersici and Ralstonia solanacearum, whose host range comprises 450 plant species (Huang and Allen 2000). Fusarium wilt (FW) and bacterial wilt (BW) of tomato, caused by these pathogens, respectively, are two very important soil-borne vascular diseases difficult to control by conventional approaches. Hence, it was felt that it was imperative to conceptualize a strategy that bolsters intrinsic response in tomato against these diseases. Previously, RRS1-R and I-3 genes have been reported to enhance the resistance of transgenic plants to pathogens (Deslandes et al. 2002; Hemming et al. 2004). The Arabidopsis RRS1-R gene, a novel single dominant disease-resistance gene, conferred resistance to R. solanacearum in transgenic Arabidopsis plants, while the tomato I-3 gene enhanced the resistance of transgenic tomato to F. oxysporum. However, because of their high host-pathogen specificity, none of these two diseaseresistance genes can confer resistance to both pathogens. In this study, an Arabidopsis thaliana thionin (Thi2.1) gene was introduced into tomato, taking care to negate transgene expression in fruits by the use of a fruit-inactive promoter (RB7) isolated from tobacco. Molecular characterization and resistance scoring of the transgenic lines demonstrated the functionality of this anti-microbial peptide against both R. solanacearum and F. oxysporum f. sp. lycopersici. Expression of Thi2.1 was incidentally observed in the leaves and roots, but not in fruits (green and ripe fruits), thereby rendering genetically modified tomatoes more palatable. Although the anti-microbial activity of Thi2.1 has been tested before (Epple et al. 1997), this is the first study to demonstrate the successful application of the Thi2.1 gene in crop plants for breeding of resistance to important fungal and bacterial diseases.
Materials and methods
(Nicotiana tabacum L. cv. W38). The primers designed to amplify the 635-bp fragment (nucleotide 635 1 from the transcription start site) were as follows: 5¢ primer (5¢-CCGGGGATCCATATGTCCTACACAATGTGAAT-3¢) and 3¢ primer (5¢-GGCCGTCGACGGTTTCCAAGTTTCACATAAC-3¢). PCR amplification was performed using DNA polymerase with proof reading activity (Promega, Madison, WI, USA) to minimize sequence mutation. The PCR product was cloned into pCAMBIA1381Z (Center for the Application of Molecular Biology of International Agriculture, Black Mountain, Australia) to form pCAMBIA1381Z-TobRB7. The Arabidopsis Thi2.1 gene was isolated by reverse transcriptase polymerase chain reaction from 3-weekold Arabidopsis leaves pretreated with 100 lM methyl jasmonate for 3 days, as described previously (Bohlmann et al. 1998). Primers designed to amplify a 418-bp DNA fragment constituting the entire Thi2.1 coding region were as follows: 5¢ primer (5¢-GTCGAC ATGAAAGGAAGAATTTTG-3¢) and the 3¢ primer (5¢-GGTGACC TTACAACAGTTTAGGC-3¢). Thi2.1 fulllength cDNA was cloned into pT7Blue (Novagen, Madison, WI, USA), and then subcloned into pCAMBIA1381Z-TobRB7 as a SalI and BstEII fragment by replacing the GUS gene, to obtain the expression vector pRB7/Thi2.1 (Fig. 1a). The cauliflower mosaic virus (CaMV35S) promoter was subcloned into pRB7/Thi2.1 to replace the RB7 promoter, thereby obtaining the constitutive expression control vector, p35S/Thi2.1 (Fig. 1a). The constructed plasmids were introduced into tomato via an Agrobacterium-mediated transformation procedure (Hsieh et al. 2002a, b). Molecular characterization of transgenic tomato plants
Tomato (L. esculentum L. Miller) cultivar CL591593D4-1-0-3 (5915) was used as the background line for transformation. Before surface sterilization, seeds were soaked at 32C for 1 h. They were treated with 1% NaOCl for 10 min and washed twice with sterile water for 5 min, and subsequently germinated on MS basal medium with a 16-h photoperiod at 26C. For BW evaluation, tomato varieties Hawaii 7996 (H7996) and L390 were used as resistant and susceptible controls, respectively (Wang et al. 2000). For FW evaluation, tomato varieties MH1 and Bonny Best were used as the resistant and susceptible controls, respectively. These seeds were kindly provided by AVRDC-The World Vegetable Center, Tainan, Taiwan.
Total genomic DNA and RNA for Southern and northern blot hybridization, respectively, was isolated from leaves of transgenic T2 plants and untransformed plants, as described previously (Hsieh et al. 2002a; Lee et al. 2003). Two primers covering the tomato ubiquitin3 gene (Ubi, X58253) coding region were used to amplify a 480-bp DNA fragment, and their sequences were as follows: 5¢ primer (5¢-GACGAAG ATGCAGATCTTCGTGAAAACCCT-3¢) and 3¢ primer (5¢-AA TCAATCGCCTCCAGCCTTGTTGTAAACG-3¢). Thi2.1, hygromycin phosphotransferase II (HptII), and Ubi cDNA labeled with (a 32P)dCTP by the random primer method were used as probes for Southern and northern blot analyses. Anti-thionin antibodies were raised and used for western blot analysis, as described previously (Epple et al. 1997).
Plasmid construction and transformation
Resistance scoring of transgenic tomato plants
The RB7 promoter (Yamamoto et al. 1991) was isolated from leaves of greenhouse-grown tobacco plants
The level of resistance of transgenic tomato plants to FW and BW was evaluated, as described previously (Lin et al.
Plant materials
388
Fig. 1 Transgene integration in transgenic tomato plants. a Binary vectors, pRB7/Thi2.1 (upper) and p35S/Thi2.1 (lower), used in this study. hptII Hygromycin phosphotransferase II cDNA sequence, PRB7 tobacco RB7 promoter, Thi2.1 Arabidopsis thionin cDNA, P35S cauliflower mosaic virus 35S promoter, Tnos nopaline synthase terminator sequence, T35S cauliflower mosaic virus 35S terminator sequence, LB left border, RB right border. The bar indicates the thionin cDNA (418 bp) used as the probe for Southern blot analysis. b Southern blot analysis. Genomic DNA (10 lg), isolated from wild-type plants (5915) and transgenic plants (R1, R3, R7, R9, and R11), was digested with SalI and subjected to the analysis with 32P-labeled Thi2.1 cDNA probe
2004). For FW tests, F. oxysporum f. sp. Lycopersici isolate Fol-34ssl (race 2) was used as the inoculum. For BW tests, R. solanacearum strain Pss4 (race 1, biovar 3) was used as the inoculum. Additionally, for R. solanacearum colonization experiments, a total of five plants were randomly harvested from each treatment at each sampling time. Plants were uprooted, washed off the soil, soaked in 70% alcohol for 3–5 min, rinsed in sterile water twice, and blotted dry on paper towels. Each plant was sectioned into the root and a 2-cm section each from the collar, the mid-stem and the top stem. Each sample was weighted, macerated, and then the internal bacterial density was measured by direct plating.
Results
in plant genome by Southern blot analysis (as depicted in Fig. 1b) and are effectively transgenic. RB7/Thi2.1 expression was confined to leaves and roots but negated in fruits Spatial transgene expression patterns were further determined in selected transgenic lines. As depicted in Fig. 2a, in transgenic lines expressing RB7/Thi2.1 (R7, R9, R11), Thi2.1 transcripts were detected only in roots and leaves but not in fruits. However, the Hph gene driven by the constitutive CaMV35S promoter was expressed in roots, leaves, and fruits in these transgenic lines. Thi2.1and Hph transcripts were not detected in wild-type plants (5915) (Fig. 2a, b), whilst they were readily detected in the root, leaves, and fruits in transgenic plants expressing 35S/Thi2.1 (35ST) (Fig. 2b). Expression of transgenes was further analyzed in fruits at two different developmental stages: green fruit (GF) and red fruit (RF). Results presented in Fig. 2c clearly indicate that Thi2.1 expression was completely negated in fruits at these two stages, while its transcripts were detected in roots of plants at the same stages. The Hph gene, however, was stably expressed in roots, GF, and RF. No Thi2.1and Hph transcripts were detected in wild-type plants.
Introduction and stable integration of Arabidopsis Thi2.1 in transgenic tomato
RB7/Thi2.1 transgenic plants exhibited enhanced resistance to soil-borne vascular wilting diseases
An expression cassette, harboring an Arabidopsis Thi2.1 gene driven by a tobacco root-specific promoter (RB7) (Fig. 1a), was introduced into tomato cultivar CL5915 by Agrobacterium tumefaciens. A total of ten putative transgenic tomato lines (T0) carrying pRB7/ Thi2.1 were obtained and initially screened by PCR. Stable transgene integration in T0 transgenic plants was confirmed by Southern blot analysis (data not shown). A few transgenic T1 lines, derived from the T0 lines displaying single-copy transgene insertions (R1, R3, R7, R9, and R11), were selected for further analyses. All five T1 lines were again confirmed for the integration of single-copy insertions of the Thi2.1 gene
Several transgenic lines were subjected to FW and BW tests, and compared to wild-type 5915 plants, in order to evaluate their resistance to these two important soilborne vascular diseases. As shown in Table 1, the T1 and T2 plants of the transgenic lines that were tested exhibited various levels of enhanced resistance to FW and BW. For FW, the transgenic lines showed significantly lower disease severity than wild-type plants, except for line R1. Particularly, lines R7 and R11 displayed low disease severity similar to the FWresistant control MH1, which possesses a single dominant resistance gene, I2 (Crill et al. 1971). For BW, the transgenic lines also showed significantly lower
389 Table 1 Evaluation of transgenic tomato plants expressing RB7/ Thi2.1 on their responses to pathogens of FW and BW T1
R1 R3 R7 R9 R11 5915 Ra Sa
T2
FWb
BWb
FWb
BWb
6.2 2.2** 0.7** 1.3** 0.5** 8.3 0.2 9.8
25.0** 60.0 20.0** 25.0** 28.0** 100.0 9.3 100.0**
5.2 1.2** 0.3** 2.1** 0.6** 9.6 0.2 9.8
33.3* 58.4 16.7** 25.0** 28.0** 100.0 10.5 100.0
a For FW evaluation, tomato varieties MH1 and Bonny Best were used as the resistant (R) and susceptible (S) controls, respectively. For BW evaluation, tomato varieties Hawaii 7996 (H7996) and L390 were used as the resistant (R) and susceptible (S) controls, respectively b Data presented were mean severity scores for FW and percent wilted plant for BW. The scoring of 5915 plants and the transgenic lines were significantly different at 0.05 (*) and 0.01 levels (**)
Fig. 2 Fruit-inactive expression of transgene Thi2.1 in transgenic plants. Total RNA (10 lg) specimens extracted from various tissues of wild-type plants (5915 in a, b, c); transgenic plants expressing RB7/Thi2.1 (R7, R9, and R11 in a, b, c) and transgenic plants expressing 35S/Thi2.1 (35ST in b) were subjected to northern blot analysis. Probes used for these experiments were 32P-labeled Thi2.1, hptII (Hph), and ubi3 (Ubi) cDNA fragments. Equal RNA loading was demonstrated by visualizing ribosomal RNA (rRNA) on a gel stained with ethidium bromide. R Root, L leaf, F fruit, GF green fruit, RF red fruit
disease severity than wild-type plants, except for line R3. However, the final disease severity of these transgenic lines was still higher than that of H7996, a tomato variety resistant to BW (Wang et al. 1998). Disease progression of bacterial wilt was delayed in RB7/Thi2.1 transgenic plants by a systemic suppression of bacterial multiplication Since BW is a very complex and serious vascular disease affecting many agronomically important crop species (Hayward 1991), the BW resistance of RB7/Thi2.1 transgenic lines was further investigated. As shown in Fig. 3, wild-type (5915) plants severely wilted 7 days
post-inoculation of R. solanacearum. Meanwhile, the BW-resistant tomato variety H7996 and RB7/Thi2.1 transgenic lines did not show obvious signs of the disease. To elucidate the defense mechanism(s) responsible for enhanced resistance to BW observed in the RB7/ Thi2.1 transgenic tomato plants, disease progression was monitored in selected transgenic lines exhibiting high levels of enhanced BW resistance (R7, R9, R11), in comparison with wild-type plants and resistant (H7996) and susceptible (L390) varieties. As represented in Fig. 4, less than 20% of the test transgenic plants and H7996 wilted during the test period, with RB7/Thi2.1 transgenic lines exhibiting an equivalent disease incidence to H7996 over the test period. In contrast, nearly 40% of the wild-type (5915) plants and 50% of L390 plants wilted only 7 days after infection, and all of them withered 35 days after infection. To further investigate the nature of the enhanced BW resistance observed in the transgenic lines, the in planta multiplication of R. solanacearum was monitored. As shown in Fig. 5, the bacterial titers in various tissues of susceptible control plants (5915 and L390) reached a very high level (=107 cfu/g fresh tissues) 7 days after inoculation, whilst the internal bacterial titers in the transgenic lines and H7996 were much lower than those in 5915 and L390. In addition, the pattern and the level of bacterial growth suppression in RB7/Thi2.1 transgenic lines were similar to that in H7996, with gradually declining levels of bacterial multiplication from roots to top stems (Fig. 5). These results revealed a systemic suppression of internal bacterial multiplication in these BW-resistant plants. R. solanacearum infection did not alter the fruit-inactive expression pattern of Thi2.1 in the transgenic lines Further studies were carried out to determine whether the fruit-inactive expression pattern of Thi2.1 could be
390 Fig. 3 RB7/Thi2.1 transgenic plants exhibited enhanced resistance to R. solanacearum. The test plants were inoculated with R. solanacearum and then kept at 28C with a photoperiod of 16 h. The photograph was taken 7 days post-inoculation. The test plants included wild-type plants (5915), a BW-resistant control variety (H7996) and T1 transgenic plants expressing RB7/Thi2.1 (R7, R9, R11)
modulated by pathogen infection. After R. solanacearum infection, transcripts of Thi2.1 remained restricted to roots and leaves in RB7/Thi2.1 transgenic plants, while Thi2.1 transcripts were detected in all of the tested tissues of 35S/Thi2.1 transgenic plants (Fig. 6a). The Thi2.1 expression pattern that was observed in these transgenic lines after pathogen infection was similar to that without pathogen infection (Fig. 2b), indicating that R. solanacearum infection did not change the fruitinactive expression pattern of the Thi2.1 in RB7/Thi2.1 transgenic plants. Results of western blot analysis showed that a protein(s) with the expected size of the transgenic Thi2.1 protein was detected in roots and leaves of RB7/Thi2.1 transgenic plants, but not in fruits post R. solanacearum infection (Fig. 6b). Transgenic Thi2.1 protein, however, was detected in the 35S/Thi2.1 line, not only in roots and leaves but also in fruits. No protein was detected in wild-type plants.
Fig. 4 Disease progression of bacterial wilt was delayed in RB7/ Thi2.1 transgenic plants. The response of the plants subjected to BW bioassays was scored as the percentage of wilted plants over time. The test plants included a resistant variety (H7996), a susceptible variety (L390), wild-type plants (5915), and transgenic plants (R7, R9 and R11). Mean comparisons were conducted over the varieties that were tested within the same recording day
Discussion In this study, we explored the potential use of thionin genes for the genetic engineering of enhanced resistance to a few destructive, soil-borne vascular diseases in tomato, including FW and BW. However, due to the general consensus that thionins are toxic to biological systems (Bussing et al. 1999), the production of transgenic tomato using thionin genes needs to be done carefully. Thus, the commonly used constitutive promoter, CaMV35S, could not be used in the present study. Instead, an organ-specific promoter was used to control the expression of the thionin transgene, Arabidopsis Thi2.1. The RB7 gene promoter, isolated form tobacco and recognized as root-specific (Conkling et al. 1990; Yamamoto et al. 1991), was employed in the present study. The expression cassette containing Thi2.1 driven by the RB7 promoter was transformed into a wilt-susceptible tomato cultivar, aiming to produce transgenic tomatoes with enhanced resistance to FW and BW but to negate Thi2.1 expression in fruits. Spatial expression analysis revealed that constitutive Thi2.1 expression in RB7/Thi2.1 transgenic plants was confined to roots and incidentally to leaves but completely negated in fruits, including GFs and RFs. It was not surprising to detect the expression of Thi2.1 in roots of RB7/Thi2.1 transgenic plants; however, the detectable Thi2.1 expression in leaves was unexpected. These results strongly indicate that, inconsistent with the previous report on transgenic tobacco plants (Yamamoto et al. 1991), the RB7 promoter was fruit-inactive in the transgenic tomato plants rather than root-specific. However, further study is required to investigate the mechanism. Western blot analysis further confirmed the potential effects and safety of expressing the thionin gene under the control of the fruit-inactive RB7 promoter. Similar approaches, using tissue-specific promoters negating the production of toxic transgene products in fruits or other consumed parts of crops, could facilitate the genetic engineering of desired traits in other crops. The functional impact of thionins on a broad spectrum of phytopathogens has been previously demon-
391
Fig. 5 R. solanacearum multiplication in RB7/Thi2.1 transgenic plants was suppressed systemically. The bacterial titer inside the test plants was measured in different tissues 7 days post-inoculation. The data are presented as the mean of the collected data. The test plants included a susceptible variety (L390), wild-type plants (5915), a resistant variety (H7996), and transgenic plants (R7, R9 and R11). Mean comparisons were conducted over tested varieties within the same recording day
strated by in vitro studies and transgenic approaches (Bohlmann et al. 1988; Carmona et al. 1993; Epple et al. 1997; Holtorf et al. 1998; Molina et al. 1993; Terras et al. 1995), but not yet extended for crop improvement. In this study, the Arabidopsis Thi2.1 gene was chosen as the target transgene for generation of transgenic tomato plants as it has been shown to be promising in fostering genetic engineering strategies for crop improvement against F. oxysporum (Epple et al. 1997). Additionally, although anti-microbial activity of Arabidopsis Thi2.1 protein against R. solanacearum has not been tested, the effect of thionins against bacterial phytopathogens has been demonstrated previously (Bohlmann et al. 1988; Carmona et al. 1993; Epple et al. 1997; Holtorf et al. 1998; Molina et al. 1993; Terras et al. 1995). Our results showed that transgenic tomato expressing RB7/Thi2.1 conferred significantly enhanced resistance against FW and BW, with the degree of enhanced resistance similar to that conferred by the corresponding resistant cultivars. These results support the effectiveness of Thi2.1 overexpression in the enhancement of resistance to FW in plants, consistent with the previous report (Epple et al. 1997). Additionally, the enhancement of resistance to BW further extends the effectiveness of Thi2.1 to a destructive and complex tomato disease. Most importantly, although the complete spectrum of enhanced disease resistance of our transgenic plants remain to be determined, the spectrum and degree of increased disease resistance observed in our transgenic lines were much more significant than that of the transgenic tomato plants reported in many previous studies (Lee et al. 2002; Li and Steffens 2002; Robison et al. 2001; Tabaeizadeh et al. 1999). Results from our study, therefore, demonstrate the safety and effectiveness of defensin/ thionin-based approaches for the genetic engineering of disease resistance in crops.
The degree and nature of enhanced resistance to BW observed in most of the tested RB7/Thi2.1 transgenic lines were similar to that in H7996, a natural BWresistant tomato cultivar. Previously, it has been demonstrated that resistance to BW in H7996 is related
Fig. 6 R. solanacearum inoculation did not alter the Thi2.1 expression pattern in transgenic plants. a Northern blot analysis. Total RNA was isolated from various tissues of the test plants infected with R. solanacearum 7 days post-inoculation and subjected to the analysis. Probes used for the analysis included 32 P-labeled Thi2.1, hptII (Hph), and ubi3 (Ubi) cDNA fragments. Equal RNA loading was demonstrated by visualizing ribosomal RNA (rRNA) on a gel stained with ethidium bromide. b Western blot analysis. Protein samples were extracted from various tissues (20 lg per lane) of the test plants 7 days post-inoculation with R. solanacearum and subjected to the analysis. The test plants included wild-type plants (5915), transgenic plants expressing RB7/ Thi2.1 (R7), and transgenic plants expressing 35S/Thi2.1 (35ST). R Root, L leaf, F fruit
392
to internal pathogen multiplication, rather than root invasion or upward movement (Wang and Lin 2003; Wang et al. 2000). Our results also indicated a similar suppression of bacterial growth in RB7/Thi2.1 transgenic lines. However, the disease severity of BW in our transgenic lines was still higher than that in H7996 (Table 1). These results suggest that the enhanced BW resistance induced by RB7/Thi2.1 overexpression in transgenic tomato may be resulted from the stimulation of either a portion of the defense system(s) responsible for the resistance conferred in H7996, or via activation of a defense pathway different from that employed by H7996. However, since the mechanism of BW resistance conferred in H7996 and the signal transduction pathway(s) involved have not been elucidated at a molecular level so far, further studies are necessary to elucidate and compare the nature of BW resistance in H7996 and that in RB7/Thi2.1 transgenic tomato plants in a more detailed way. In summary, the present study demonstrates that a toxic protein with anti-microbial activity, Arabidopsis Thi2.1, can be successfully used for the genetic engineering of enhanced resistance in tomato to important fungal and bacterial pathogens, without expressing potentially toxic compounds in fruits. The employment of the novel fruit-inactive RB7 promoter for crop improvement by genetic engineering approaches could be particularly important and desirable due to public concerns about the safety of genetically modified fruit crops. To the best of our knowledge, this is the first study using Arabidopsis Thi2.1 to confer pathogen resistance in crop plants, with the potentially toxic products restricted to non-consumed parts of the crop, thus making a great breakthrough in the crop improvement process. In addition, this study also manifests that the transgenic plants exhibited a high degree of resistance to the major diseases in tomato, specifically FW and BW, which is also a notable improvement in the breeding of tomato cultivars. Similar strategies may also aid other important crop breeding programs for disease resistance, potentially against a spectrum of pathogens. Acknowledgements We are grateful to The Institute of Molecular Biology for providing experimental equipments and facility and AVRDC—The World Vegetable Center for their technical assistance. We also thank Dr. Kenrick Deen for his critical review of this manuscript. This work was supported by a grant from Academia Sinica and grant NSC-92-2317-B-001-037 from the National Science Council of the Republic of China. This work was carried out in compliance with the current laws governing genetic experimentation in Taiwan, the Republic of China. YuanLi Chan and Venkatesh Prasad contributed equally to this work. Chiu-Ping Cheng and Ming-Tsair Chan also contributed equally to this work.
References Bohlmann H (1994) The role of thionins in plant protection. Crit Rev Plant Sci 13:1–6
Bohlmann H, Clausen S, Behnke S, Giese H, Hiller C, Schrader G, Barkholt V, Apel K (1988) Leaf-thionins of barley—a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defense mechanism of plants. EMBO J 7:1559– 1565 Bohlmann H, Vignutelli A, Hilpert B, Miersch O, Wasternack C, Apel K (1998) Wounding and chemicals induce expression of the Arabidopsis thaliana gene Thi2.1, encoding a fungal defense thionin, via the octadecanoid pathway. FEBS Lett 437:281–286 Bussing A, Stein GM, Wagner M, Wagner B, Schaller G, Pfuller U, Schietzel M (1999) Accidental cell death and generation of reactive oxygen intermediates in human lymphocytes induced by thionins from Viscum album L. Eur J Biochem 262:79–87 Carmona MJ, Molina A, Fernandez JA, Lopez-Fando JJ, GarciaOlmedo F (1993) Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J 3:457–462 Conkling M, Cheng C-L, Yamamoto Y, Goodman H (1990) Isolation of transcriptionally regulated root-specific genes from tobacco. Plant Physiol 93:1203–1211 Crill P, Strobel J, Burgis D, Bryan H, John C, Everett P, Bartz J, Hayslip N, Deen W (1971) Florida MH-1, Florida’s first machine harvest fresh market tomato. Agricultural Experiment Station, University of Florida, p 12, Circular S-212 Deslandes L, Olivier J, Theulieres F, Hirsch J, Feng DX, BittnerEddy P, Beynon J, Marco Y (2002) Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proc Natl Acad Sci USA 99:2404–2409 Epple P, Apel K, Bohlmann H (1997) Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell 9:509–520 Epple P, Vignutelli A, Apel K, Bohlmann H (1998) Differential induction of the Arabidopsis thaliana Thi2.1 gene by Fusarium oxysporum f. sp. matthiolae. Mol Plant Microbe Interact 11:523– 239 Fernandez de Caleya R, Gonzalez-Pascual B, Garcia-Olmedo F, Carbonero P (1972) Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro. Appl Microbiol 23:998–1000 Hayward A (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29:65–87 Hemming MN, Basuki S, McGrath DJ, Carroll BJ, Jones DA (2004) Fine mapping of the tomato I-3 gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for I-3. Theor Appl Genet (in press) Holtorf S, Ludwig-Muller J, Apel K, Bohlmann H (1998) Highlevel expression of a viscotoxin in Arabidopsis thaliana gives enhanced resistance against Plasmodiophora brassicae. Plant Mol Biol 36:673–680 Hsieh TH, Lee JT, Charng YY, Chan MT (2002a) Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol 130:618–626 Hsieh TH, Lee JT, Yang PT, Chiu LH, Charng YY, Wang YC, Chan MT (2002b) Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol 129:1086–1094 Huang Q, Allen C (2000) Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants. Physiol Mol Plant Pathol 57:77–83 Lee T, Coyne D, Clemente T, Mitra A (2002) Partial resistance to bacterial wilt in transgenic tomato plants expressing antibacterial Lactoferrin gene. J Am Soc Hortic Sci 127:158–164 Lee J-T, Prasad V, Yang P-T, Wu J-F, Ho DT-H, Charng Y-Y, Chan M-T (2003) Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield. Plant Cell Environ 26:1181–1190 Li L, Steffens J (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247
393 Lin W-C, Lu C-F, Wu J-W, Cheng M-L, Lin Y-M, Yang N-S, Black L, Green SK, Wang J-F, Cheng C-P (2004) Transgenic tomato plants expressing the Arabidopsis NPR1 gene confer enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Res 13:(in press) Molina A, Ahl-Goy P, Fraile A, Sanchez-Monge R, GarciaOlmedo F (1993) Inhibition of bacterial and fungal plant pathogens by thionins of types I and II. Plant Sci 92:169–177 Robison M, Shah S, Tamot B, Pauls K, Moffatt B, Glick B (2001) Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase. Mol Plant Pathol 2:135– 145 Rommens CM, Kishore GM (2000) Exploiting the full potential of disease-resistance genes for agricultural use. Curr Opin Biotechnol 11:120–125 Tabaeizadeh Z, Agharbaoui Z, Harrak H, Poysa V (1999) Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2. Plant Cell Rep 19:197–202 Terras FRG, Eggermont K, Kovaleva V, Raikhel NV, Osborn RW, Kester A, Rees SB, Torrekens S, Leuven FV, Vanderleyden J,
Cammue BPA, Broekaert WF (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573–588 Wang JF, Lin CH (2003) Colonization capacity of Ralstonia solanacearum tomato strains differing in aggressiveness on tomatoes and weeds. In: Allen CPP, Hayward AC (eds) Bacterial wilt: the disease and the Ralstonia solanaceraum species complex. APS, St Paul (in press) Wang J, Hanson P, Barnes J (1998) Worldwide evaluation of an international set of resistance sources to bacterial wilt in tomato. In: Prior P, Allen C, Elphinstone J (eds) Bacterial wilt disease. Molecular and ecological aspects. Springer, Berlin Heidelberg New York, pp 269–275 Wang JF, Olivier J, Thoquet P, Mangin B, Sauviac L, Grimsley NH (2000) Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus. Mol Plant Microbe Interact 13:6–13 Yamamoto Y, Taylor C, Acedo G, Cheng C, Conkling M (1991) Characterization of cis-acting sequences regulating root-specific gene expression in tobacco. Plant Cell 3:371–382
