Transgenic Research 13: 567–581, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases Wan-Chi Lin1, Ching-Fang Lu1, Jia-Wei Wu1, Ming-Lung Cheng1, Yu-Mei Lin1, Ning-Sun Yang, Lowell Black2, Sylvia K. Green2, Jaw-Fen Wang2 & Chiu-Ping Cheng1,* 1 2
Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan 115, R. O. C. AVRDC-The World Vegetable Center, P.O. Box 42, Shanhua, Tainan, Taiwan 741, R. O. C.
Received 26 August 2003; accepted 16 June 2004
Key words: Arabidopsis NPR1, disease resistance, PR genes, tomato
Abstract Development of eﬀective disease-resistance to a broad-range of pathogens in crops usually requires tremendous resources and eﬀort when traditional breeding approaches are taken. Genetic engineering of disease-resistance in crops has become popular and valuable in terms of cost and eﬃcacy. Due to longlasting and broad-spectrum of eﬀectiveness against pathogens, employment of systemic acquired resistance (SAR) for the genetic engineering of crop disease-resistance is of particular interest. In this report, we explored the potential of using SAR-related genes for the genetic engineering of enhanced resistance to multiple diseases in tomato. The Arabidopsis NPR1 (nonexpresser of PR genes) gene was introduced into a tomato cultivar, which possesses heat-tolerance and resistance to tomato mosaic virus (ToMV). The transgenic lines expressing NPR1 were normal as regards overall morphology and horticultural traits for at least four generations. Disease screens against eight important tropical diseases revealed that, in addition to the innate ToMV-resistance, the tested transgenic lines conferred signiﬁcant level of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW), and moderate degree of enhanced resistance to gray leaf spot (GLS) and bacterial spot (BS). Transgenic lines that accumulated higher levels of NPR1 proteins exhibited higher levels and a broader spectrum of enhanced resistance to the diseases, and enhanced disease-resistance was stably inherited. The spectrum and degree of these NPR1transgenic lines are more signiﬁcant compared to that of transgenic tomatoes reported to date. These transgenic lines may be further explored as future tomato stocks, aiming at building up resistance to a broader spectrum of diseases.
Introduction Genetic engineering of disease-resistance through transferal of plant defense-related genes or pathogen-originated genes into crops is valuable in terms of cost, eﬃcacy and reduction of pesticide usage (Shah, 1997; Salmeron & Vernooij, 1998; Rommens & Kishore, 2000; Stuiver & Custers, 2001). Among the strategies used for the genetic *Author for correspondence E-mail: [email protected]
engineering of disease-resistance, the employment of SAR is of special interest. In an incompatible plant-pathogen interaction, in addition to the immediate activation of a whole complex defense network and hypersensitive response blocking pathogen growth at the site of initial infection, SAR is established in uninfected parts of the plant for further resistance to a broad-spectrum of pathogens (Ryals et al., 1996). SAR is long lasting and often associated with local and systemic accumulation of salicylic acid (SA) (Malamy et al., 1990; Me´traux et al., 1990; Rasmussen et al.,
568 1991) and induced expression of a number of genes, including a group of pathogenesis-related (PR) genes (Ward et al., 1991; Ryals et al., 1996). Overexpression of some PR genes in transgenic plants conferred modest protection against pathogens (Broglie et al., 1991; Alexander et al., 1993; Liu et al., 1994; Zhu et al., 1994; Jach et al., 1995). However, the protection provided by a single speciﬁc PR gene is usually very limited in its spectrum, degree and duration, compared to that of a native SAR response (Jach et al., 1995; Jongedijk et al., 1995). Therefore, more durable resistance to a broader spectrum of pathogens may be expected from engineering defense reactions that are more closely related to the natural SAR defense mechanisms employed in most incompatible plant-pathogen interactions. To elucidate the mechanism of SAR and identify important genes involved in this response, extensive eﬀorts were spent on genetic screens of Arabidopsis mutants (Clarke et al., 1998). Particularly, screens of Arabidopsis mutants defective in response to the SA signal have led to identiﬁcation of NPR1 (also known as NIM1 or SAI1) (Cao et al., 1994; Delaney et al., 1995; Glazebrook et al., 1996; Shah et al., 1997). NPR1 encodes a protein that contains multiple ankyrin repeat domains that is found in some regulatory proteins (Cao et al., 1997; Ryals et al., 1997). The NPR1 gene product is localized in the nucleus (Kinkema et al., 2000) where it physically and diﬀerentially interacts with Arabidopsis TGA family members of basic region, leucine zipper (bZIP) transcription factors (Zhang et al., 1999; Despre´s et al., 2000; Zhou et al., 2000; Subramaniam et al., 2001; Fan & Dong, 2002; Kim & Delaney, 2002). Under normal conditions, NPR1 expresses at a very low level; however, upon treatment of plants with SA, 2,6-dichloroisonicotinic acid (INA) or benzo [1,2,3] thiadiazole-7carbothioic acid S-methyl ester (BTH), the level of NPR1 gene expression is enhanced (Cao et al., 1997; Ryals et al., 1997), and regulated through SA-induced WRKY DNA binding proteins (Yu et al., 2001) and redox changes (Mou et al., 2003). In fact, it has been shown that priming of defense gene activation by chemical SAR inducers requires NPR1 (Conrath et al., 2002; Kohler et al., 2002). In addition, NPR1 is also involved in jasmonic acid (JA)/ethylene-dependent induced systemic resistance (ISR) shared by some
Pseudomonas ﬂourescens strains (Pieterse et al., 1998), and has been shown to modulate cross talk between salicylate- and jasmonate-dependent defense pathways (Spoel et al., 2003). Therefore, the NPR1 protein is a key regulator in the transduction of SA and JA/ethylene signals leading to general acquired resistance responses. Overexpression of NPR1 in Arabidopsis has been shown to lead to a non-speciﬁc resistance to Peronospora parasitica and Pseudomonas syringae pv. maculicola in a dosage-dependent fashion, without obvious detrimental eﬀects on the plants (Cao et al., 1998). Overexpression of NPR1 also results in enhanced eﬃcacy of fungicides in Arabidopsis (Friedrich et al., 2001). In addition, transgenic rice plants overexpressing the Arabidopsis NPR1 gene exhibit enhanced resistance to the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Chern et al., 2001). A disease-resistance pathway similar to that of the Arabidopsis NPR1-mediated signaling pathway was demonstrated in rice, suggesting a conserved signal transduction pathway controlling NPR1-mediated resistance shared by monocot and dicot plants (Zhang et al., 1999; Chern et al., 2001). In addition to its key role in activating SAR and ISR, NPR1 has also been suggested to be involved in regulating cell growth (Vanacker et al., 2001). Tomato is an economically important crop worldwide, and is susceptible to various fungal, bacterial, viral and nematode diseases, particularly in the tropics. Resistance to multiple diseases is an important trait when developing tropically adapted varieties of tomato. However, such task requires tremendous resources and eﬀorts when traditional breeding approaches by crossing are taken. Therefore, this study explored the potential of employing SAR-related genes for the genetic engineering of long lasting and broad-range disease-resistance in tomato. Here, we report the generation and characterization of transgenic tomato plants overexpressing Arabidopsis NPR1. The transgenic lines generated from this study exhibited signiﬁcant enhancement of resistance to important bacterial and fungal diseases, without causing deleterious eﬀects on plant growth and development. Importantly, the spectrum and degree of these transgenic lines are more signiﬁcant compared to that of transgenic tomatoes reported to date. These transgenic lines
569 could be useful tomato stocks for breeding further resistance to more diseases, or used as resource for breeding resistance to BW and FW in other tomato varieties.
Materials and methods Transgene construction and plant transformation The NPR1 coding sequence was isolated from Arabidopsis thaliana ecotype Col0 by polymerase chain reaction (PCR) using primers designed based on the published sequence (Cao et al., 1997; Ryals et al., 1997) and cloned under the control of the 35S promoter of Cauliﬂower Mosaic Virus (CaMV 35S promoter). This transgene cassette was further cloned into the binary Ti-plasmid vector pCAMBIA1301 (Center for the Application of Molecular Biology to International Agriculture, CAMBIA, Inc., Canberra, ACT, Australia), which carries kanamycin resistance gene and hygromycin resistance gene (hygromycin phosphotransferase) as bacterial selection and plant transformation selection genes, respectively. The construct was transformed into the Agrobacterium tumefaciens strain LBA4404 by cold-shock transformation (An, 1987). Agrobacterium-mediated transformation was performed on cotyledon explants of 7–8 day old seedlings of Lycopersicon esculentum according to Fillatti et al. (1987). Hygromycin at 20 mg/l in culture medium was used to select transformants (deﬁned as R0 lines), followed by staining for a GUS activity. The tomato line used for the transformation was CL5915-93D4-1-0-3 (CL5915), a line bred at the AVRDC-The World Vegetable Center, Taiwan. This tomato line is a small-fruit, highly heat-tolerant line that has been cultivated in several tropical countries. It has been used as a good parental line for generating heat tolerant hybrid cultivar for tropical and subtropical areas. R1 lines were progeny lines deriving from the R0 transgenic lines and R2 lines were progeny lines deriving from the R1 transgenic lines. Molecular characterization of transgenic plants For genomic Southern blot analysis, genomic DNA was isolated from plant leaves using a CTAB method described by Saghai-Maroof et al.
(1984). Five micrograms of genomic DNA digested with the appropriate restriction enzymes was separated in 0.8% agarose gels in 1X TBE buﬀer and then transferred to NYTRAN membranes (Schleicher & Schuell BioScience, Keene, NH, USA) following a procedure described by Sambrook and Russell (2001). EcoRI and Bam HI are restriction enzymes that cut only once in the NPR1 expression binary vector. For northern blot analysis, total RNA was extracted from leaves using TRIzol Reagent (Invitrogen Life Technologies, Co., Carlsbad, CA, USA). Five micrograms of total RNA was separated in 1.0% agarose gels containing 6.6% formaldehyde and then transferred to positively charged Nylon Membranes (Roche Molecular Biochemicals, Mannheim, Germany). The probes used for Southern and northern blot analyses were labeled with digoxigenin (DIG), following manufacture’s instructions (Roche Molecular Biochemicals). The blots were probed with the appropriate labeled probes at 42C overnight, and then subjected to 15 min washes in 2X SSC containing 0.1% SDS at room temperature twice, and in 0.5X SSC containing 0.1% SDS at 68C twice. PR1b1 (Y08804), PR1p6 (M69248), acidic b-1,3-glucanase (GLUa, M80604), basic b-1,3-glucanase (GLUb, M80608), acidic chitinase 9 (CHI9, Z15140) and basic chitinase 3 (CHI3, Z15141) were used as probes to analyze the expression of defense-related genes in the transgenic plants by northern blot analysis. In addition, reverse transcription PCR (RT-PCR) was performed for the detection of transgene expression using ReadyTo-Go RT-PCR Beads (Amersham Pharmacia Biotech, Inc., Piscataway). For western blot analysis, total proteins were extracted from plant leaves as described by Jinn et al. (1993), and 20 lg of the total proteins was subjected to SDSPAGE electrophoresis. After transfer to PVDF membranes (Millipore Co., Billerica, MA, USA), the immunoblots were developed with an antiserum raised against NPR1 recombinant proteins, following the procedure described in the manual ‘‘Immuno-Blot Assay Procedure’’ (Bio-Rad Laboratories Inc., Hercules, CA, USA). Since we failed to express full-length NPR1 protein in E. coli cells, the carboxy-terminal half (310 amino acids) and the amino-terminal half (300 amino acids) of NPR1 protein were expressed as recombinant proteins in E. coli individually by cloning
570 the corresponding sequences into the pET28a vector (Novagen, Madison, WI, USA). These two recombinant proteins were then puriﬁed from IPTG-induced E. coli cells following manufacture’s instruction, mixed at a ratio of 1:1 and used to immunize rabbits for the production of an antiserum. Disease screens of transgenic plants Several important tropical diseases of tomato were selected as targets for evaluation of resistance of transgenic lines in the disease screens. The fungal diseases selected for the screens were Fusarium wilt (FW, caused by Fusarium oxysporum f. sp. lycopersici), gray leaf spot (GLS, caused by Stemphylium solani), and late blight (LB, caused by Phytophthora infestans). The selected bacterial diseases were bacterial spot (BS, caused by Xanthomonas campestris pv. vesicatoria) and bacterial wilt (BW, caused by Ralstonia solanacearum). Viral diseases caused by Cucumber Mosaic Virus (CMV), ToMV, and Tomato Yellow Leaf Curl Virus (TYLCV) were included for the disease screens. The evaluation protocols have been routinely used at the Asian Vegetable Research and Development Center (AVRDC). Tomato varieties resistant and susceptible to each disease were included in each evaluation as controls to ensure reliability of the results. Generally seedlings were raised in 2 inch pots with pasteurized potting mixture, except plants to be tested for FW where seedlings were grown in plastic trays with cell size of 4 cm · 4 cm. Evaluation protocols are described below. For all the evaluations, plants were kept in a greenhouse at a mean temperature of 28C, unless mentioned otherwise. All evaluations were conducted at the R1 and R2 stage. Some of the R2 lines were derived from individual plants displaying enhanced resistance at the R1 stage. For FW evaluation, Isolate Fol-34ssl (race 2) was used to prepare inoculum. The entire contents of 7-day-old potato dextrose agar cultures were blended with 125 ml of distilled water per plate for 10 s. The slurry contained about 107 conidia/ml. Tomato seedlings were uprooted and immersed in the slurry for 5 min, transplanted to individual cells in plastic trays, and kept in a greenhouse at a mean temperature of 25C. Tomato variety MH-1 and Bonny Best
were used as resistant and susceptible controls, respectively. In total, 18 plants per line were tested in a complete randomized design (CRD). The severity score was rated 21 days post inoculation with 0 – no symptoms, 5 – stunted and vascular browning, 7.5 – severely stunted and vascular browning, and 10 – wilted beyond recovery. For LB evaluation, sporangium suspension (5 · 104 sporangia/ml) of Isolate Pi39A was prepared from 12 to 14 day-old rye A agar cultures and chilled at 12C for 2–3 h for zoospore release. Leaves of 5-week-old tomato seedlings were sprayed with the chilled suspension until run-oﬀ. A total of 24 plants per line were tested in a CRD. Tomato varieties L3708 and TS19 were used as resistant and susceptible controls, respectively. Inoculated plants were kept at 20C and rated 7–10 days after inoculation. The severity scores ranged from 0 to 6, where 0 means no symptoms and 6 means 91–100% leaf area aﬀected and/or extensive stem damage or plant dead. For GLS evaluation, conidia were harvested from 7-day-old V8 cultures and made into suspension (2.5 · 104 conidia/ml). Leaves of 5-week-old tomato seedlings were sprayed with the conidium suspension until run-oﬀ. A total of 24 plants per line were tested in a CRD. Tomato varieties UC82-L and Bonny Best were used as the resistant and susceptible controls, respectively. Inoculated plants were scored 7 days after inoculation with levels 0–4, where 0 means no symptoms, 1 means very few small lesions, 2 means about 10 lesions per leaf, 3 means numerous lesions per leaf, and 4 means coalescing lesions and leaf collapse. For BW evaluation, suspensions of Strain Pss4 (race 1, biovar 3) were prepared to give an OD600 equal to 0.3 (about 108 cfu/ml) and poured over the pot soil surface of 3-week-old seedlings as described previously (Wang et al., 2000). A randomized complete block design (RCBD) was used with three replications and 12 plants per replication. Tomato cultivars Hawaii7996 (H7996) and L390 were used as the resistant and susceptible controls, respectively. The response of the inoculated plants was evaluated as the percentage of wilted plants 28 days after inoculation. For BS evaluation, suspension of Strain XVP28 (race P1) was prepared from overnight 523 cultures to give an OD600 equal to 0.3 (about
571 108 cfu/ml). Prior to inoculation, 0.025% (v/v) of Silwet L-77 surfactant was added to the bacterial suspension. The entire upper ground part of 3-week-old seedlings were dipped into the suspension for 30 s. Tomato cultivars Hawaii 7998 and Bonny Best were used as the resistant and susceptible controls, respectively. A total of 12 plants per line were tested in a CRD. The response of the inoculated plants was evaluated with BarratHorsfall scores 10–12 days post inoculation. Score 0 means no symptom development; score 1 means less than 3% of leaf area showing symptoms; score 2 means 3–6% of leaf area showing symptoms; score 3 means 3–6% of leaf area showing symptoms; score 4 means 3–6% of leaf area showing symptoms. For ToMV evaluation, the inoculum was prepared by grinding leaves of ToMV-infected Nicotiana tabacum Samsun in 0.03 M Na-phosphate buﬀer (pH 7.0) amended with 0.5% (w/v) Na2SO3, 0.17% (w/v) Na2-DIECA and 0.01% (w/v) charcoal. The leaf to buﬀer ratio (w/v) was 1 g to 4 ml. Tomato seedlings were inoculated at the 3–4-leaf stage by spreading the inoculum on leaves in the presence of carborundum. ELISA tests were carried out two weeks after inoculation to detect the virus in the inoculated plants. Tomato variety TK70 was used as the susceptible control. A total of 24 plants per line were tested in a CRD. For CMV evaluation, the inoculum was prepared by grinding leaves of CMV-infected Nicotiana glutinosa in the same buﬀer and ratio as for ToMV. Seedling inoculation was conducted as above, except that a second inoculation was conducted one week after the ﬁrst inoculation. ELISA tests were carried out two weeks after the second inoculation to detect the virus in the inoculated plants. Tomato variety TK70 was used as the susceptible control. A total of 24 plants per line were tested in a CRD. For TYLCV evaluation, tomato seedlings at the 1–2-leaf stage were exposed to viruliferous whiteﬂies (Bemisia tabaci). Visual symptoms were recorded once a week until clear symptoms appeared on all plants of the susceptible control (normally 1–1.5 months). Tomato variety TK70 was used as the susceptible control. To conﬁrm visual symptom observations, all plants were subjected to nucleic acid hybridizations with a 1.4 kb probe of the TYLCV-TW (GenBank No.
U88692) (Shih et al., 1995). A total of 24 plants per line were tested in a CRD. To conﬁrm the enhanced resistance to BW, an R2 (26–36, derived from R1 Line 26) and an R3 (9-BW54-1, derived from R2 Line 9-BW54) line were selected for a time-course study. The inoculation method and experiment design were conducted as described above, except the percent wilted plants were recorded every 3–7 days until 28 days after inoculation. R2 Lines 26–36 and R3 Line 9-BW94-BW8 (derived from R2 Line 9-BW54) were used to determine the colonization degree by Fusarium oxysorum f.sp. lycopersici over time. RCBD was used with 40 plants per replication, and the inoculation was performed as described above. Stem segments (about 0.2 cm in length) were removed from collar and mid-stem (mid-point of stem) regions at 3, 7, 10, 14, and 21 days after inoculation. A total of ﬁve plants were randomly selected from each replication at each time point. Each segment was split into four sections. Pathogen colonization frequency was determined as the percentage of colonized sections, in which the result of isolating FW pathogen from the stem was positive. Two statistical methods were used to check whether the transgenic lines performed diﬀerently from the wild-type plants, CL5915, or other control varieties. For BW screening and the FW colonization experiment, the percentage data was transformed by arcsin square root for ANOVA and mean comparison by LSD. For the other diseases with rating scores, the Mann Whitney non-parametric U test was conducted to test the null hypothesis that the ratio of CL5915 (wildtype) plants across the rating scores was equal to the ratio observed for the transgenic lines.
Results Generation and identiﬁcation of stable tomato transgenic lines To express Arabidopsis NPR1 in tomato, a transgene expression plasmid was constructed, in which the NPR1 coding region was driven by a CaMV 35S promoter and gusA was used as the reporter gene. This plasmid was introduced into a tomato line, namely CL5915-93D4-1-0-3 (CL5915), via an Agrobacterium-mediated transformation method.
572 The putative transgenic plants were ﬁrst screened by hygromycin resistance selection, and then by GUS activity assay and genomic PCR (data not shown). Integration of the transgene into the genome of transgenic plants was further conﬁrmed by genomic Southern blot analysis in eight of the transgenic lines, with the results showing that the tested transgenic lines all contained a single copy of the transgene in their genome, except Line 46 (Figure 1). GUS activity assay and genomic PCR were further used to analyze the transgene segregation rate in the R1 generation, and to determine the homozygosity in the R2 generation (data not shown). The overall horticultural traits and development of these transgenic lines were similar to wild-type plants for over four generations (from R0 to R3) under regular greenhouse conditions.
transgenic lines could be classiﬁed into three groups: three high transgene expressers, Lines 9, 26, and 27; one medium transgene expresser, Line 49; and three low transgene expressers, Lines 14, 46 and 48. Western blot analysis, using an antiserum raised against NPR1, detected a protein with the expected size of the transgenic protein in all transgenic lines, except Line 14, but not in wild-type plants (Figure 2(b)). In addition to the expected protein, other proteins were also detected by this antiserum. Further analysis, using an aﬃnity-puriﬁed antibody prepared
Transgene expression in transgenic lines Transgene expression in transgenic lines was analyzed in R1 and R2 plants by northern and western blot analysis, as shown in Figures 3 and 4. Transgenic transcripts were detected in all R1 transgenic lines at varied levels by northern blot or RT-PCR analysis, but transgenic transcripts were not detected in wild-type plants (Figure 2(a)). Based on the transgenic transcript levels detected by northern blot analysis, the
Figure 1. Southern blot analysis of transgenic plants. Genomic DNA (5 lg) isolated from wild-type plants and diﬀerent transgenic lines was digested with Bam HI (top panel) or Eco RI (bottom panel) and hybridized with the DIG-labeled NPR1 cDNA probe. Eco RI and Bam HI are the restriction enzymes that cut only once in the NPR1 expression binary vector. WT: wild-type CL5915; 9, 14, 26, 27, 46, 48, 49: transgenic lines in the CL5915 background.
Figure 2. Expression of NPR1 and PR genes in R1 transgenic plants. (a) Northern blot analysis. Blots containing total RNA (5 lg), isolated from wild-type CL5915 plants (WT) and diﬀerent R1 transgenic lines (9, 14, 26, 27, 46, 48, 49), were probed with the DIG-labeled NPR1 cDNA probe. (b) Western blot analysis. Total protein extracts (20 lg), isolated from wild-type plants and diﬀerent R1 transgenic lines, were electrophoresed, transferred to membranes and blotted with an antiserum raised against NPR1 recombinant proteins. The arrow indicates the transgenic NPR1 protein expressed in transgenic plants, but not in wild-type plants. (c) PR gene expression in transgenic lines. RNA blots were probed with PR1b1 (Y08804), PR1p6 (M69248), acidic b-1,3-glucanase (GLUa, M80604), basic b-1,3-glucanase (GLUb, M80608), acidic chitinase 9 (CHI9, Z15140) and basic chitinase 3 (CHI3, Z15141). The ethidium bromide-stained 28S rRNA in the gels used for northern blot analysis is shown to demonstrate equal loading of RNA.
Figure 4. Disease progress of tomato lines after inoculation with R. solanacearum Pss4. Hawaii 7996 (H7996) is a tomato line resistant to bacterial wilt. CL5915 is the wild-type plant. 9-BW54-1 and 26–36 are the R3 and R2 lines derived from CL5915, respectively, constitutively expressing the NPR1 gene. Mean comparisons were conducted over tested varieties within the same recording day. Results of the mean comparison were only presented for the days when signiﬁcant diﬀerence was identiﬁed. Data points marked with the same letters were not signiﬁcantly diﬀerent according to Duncan’s multiple-range test (P ¼ 0.05).
gene expression was heritable at transcriptional level, but that transgene silencing may have occured in some individuals. Figure 3. Expression of NPR1 and PR genes in R2 transgenic plants. (a) Northern blot analysis. (b) Western blot analysis. (c) PR gene expression in transgenic lines. Northern and western blot analyses were performed as described in Figure 2.
against the NPR1 recombinant proteins, indicated that some of the detected proteins may be proteins derived from the transgenic proteins, while some may be endogenous gene products of NPR1-related homologue(s) (data not shown). In general, the amounts of the transgenic proteins accumulated in individual R1 lines were consistent with the levels of transgenic transcripts (Figure 2(b)). The patterns of transgene expression in R2 homozygous plants deriving from three R1 lines were also consistent with that in the parent R1 lines at the transcriptional level (Figure 3(a)). R2 lines deriving from high transgene expressers (Lines 9 and 26) accumulated high levels of transcripts, whereas the transcript levels in R2 lines deriving from the low transgene expresser (Line 14) were too low to be detected by northern blot analysis. However, despite the levels of transgenic transcripts being similar among cognate R2 lines, the amounts of transgenic proteins varied (Figure 3(b)). These results indicated that trans-
Defense gene expression in transgenic lines As induction of NPR1 expression leads to the activation of a group of PR genes (Cao et al., 1998), we further assessed expression of several tomato PR genes in R1 and R2 transgenic plants by northern blot analysis (Figures 2(c) and 3(c)). Results showed that these PR genes were constitutively expressed at diﬀerent levels in wild-type plants. In R1 transgenic lines, although the expression patterns of these test PR genes did not apparently correlate with the level of transgene expression, higher levels of transgene expression in some of the high transgene expressers (i.e. lines 26 and 27) led to more signiﬁcant degree of enhanced expression of a few PR genes (e.g., GLUa, GLUb and CHI3) (Figure 2(c)). However, no clear correlation between PR gene expression and transgene expression was observed in R2 transgenic lines (Figure 3(c)). Disease screens of transgenic lines For future cultivation of the promising transgenic tomato plants in the tropics, we evaluated
574 resistance of the transgenic lines to eight important tropical diseases. The diseases selected for this evaluation included leaf-localized, systemic and vascular diseases caused by fungal, bacterial and viral pathogens. Seven transgenic lines at R1 stage and three transgenic lines (derived from lines 9, 14 and 26) at R2 stage were subjected to disease screens to determine whether the transgenic lines displayed increased levels of resistance to the chosen diseases compared to wild-type CL5915 plants. Tomato CL5915 innately processes resistance to ToMV, and all of the tested R1 and R2 transgenic lines remained resistant to ToMV (data not shown). When challenged with TYLCV, CMV and the pathogens of LB, all R1 lines exhibited susceptibility similar to wild-type plants (data not shown). However, R1 transgenic lines exhibited various levels of enhanced resistance to FW, BW, BS and GLS. Particularly, signiﬁcant NPR1induced enhancement of resistance to FW and BW was observed (Table 1). For FW, the three
high NPR1-expressers (lines 9, 26, and 27) showed lower disease severity than wild-type plants. Particularly, lines 26 and 27 displayed low disease severity symptoms similar to the FW-resistant control MH-1, which processes a single dominant resistance gene, I2 (Crill et al., 1971). Transgenic lines with medium or low levels of NPR1 expression, however, showed similar or slightly higher severity than wild-type plants. These data suggest that enhanced FW-resistance may require a threshold level of NPR1 expression in transgenic tomato plants. For BW, R1 lines 9, 14, 26 and 49 showed signiﬁcantly lower disease severity symptoms than wild-type plants (Table 1). However, the ﬁnal disease severity symptoms of these transgenic lines was still higher than that of H7996, a tomato cultivar resistant to BW (Wang et al., 1998). In addition, enhanced resistance to BW observed in R1 transgenic lines did not correlate with the level of transgene expression. For instance, among the high NPR1-expressers, lines 9 and 26 displayed enhanced resistance to BW,
Table 1. Evaluation of transgenic tomato plants expressing NPR1 on their responses to FW, GLS, BW and BS pathogens. Data presented are maximum severity symptoms of each disease R1 lines
9 14 26 27 46 48 49 Ra Sa
2.2/7.6** 7.1/7.6ns 0.6/7.2** 0.3/7.2** 8.3/6.5** 6.4/6.5ns 6.5/6.5ns 0.0 9.4
50.0/91.7* 16.7/91.7* 33.3/100.0* 75.0/100.0ns 66.7/91.7ns 100.0/91.7ns 33.3/91.7* 11.3 100.0
9–11 9–34 9-FW51 9-BW54 14–15 14–25 14–31 14–32 14-BW54 26–36 26-FW51 26-FW52 26-BW54 CL5915a Ra Sa
3.2** 2.1** 3.0** 1.0** 3.9* 2.8** 5.2ns 2.6** 3.2** 0.6** 2.5** 2.6** 3.8* 5.7 0.2 8.5
3.0** 2.8** 2.6** 2.4** 3.4** 3.8ns 3.7ns 2.9** 3.2** 3.2** 3.4** 3.3** 3.5** 3.9 0.4 4.0
25.0** 58.3ns 33.3* 25.0** 41.7ns 41.7ns 50.0ns 41.7ns 25.0** 25.0** 25.0** 33.3* 58.4ns 58.3 8.3 100.0
33.3** n.t.d 25.0** 33.3**. n.t. n.t. n.t. n.t. 29.2** 29.2** 58.3** 33.3** 45.8** 95.8 12.5 100.0
3.2ns 3.5ns 2.3** 2.7ns 3.2ns 3.2ns 3.3ns 2.7ns 3.2ns 3.2ns 3.3ns 2.8ns 3.0ns 3.3 1.0 3.3
a Varieties resistant (R) and susceptible (S) to each disease were used as controls. They were MH1 (R) and Bonny Best (S) for FW, UC82-L (R) and Bonny Best (S) for GLS, Hawaii 7996 (R) and L390 (S) for BW, and Hawaii 7998 (R) and Bonny Best (S) for BS. Data of the control varieties in the R1 evaluations were mean over two or three trials. b Data presented were mean severity scores. Results of the Mann Whitney U non-parametric test for testing the null hypothesis that the ratio of plants of CL5915 across the rating scores is equal to the ratio observed for the transgenic lines were marked as non-signiﬁcant (ns) and signiﬁcantly diﬀerent at 0.05 (*) and 0.01 level (**). Comparison of each R1 line was made with CL5915 in the speciﬁc trial. c Data presented were mean percent wilted plant. The percentage data was transformed with arcsin square root for ANOVA and the results of mean comparison by LSD were marked as non-signiﬁcant (ns) and signiﬁcantly diﬀerent at 0.05 (*) or 0.01 (**) level. For the BW2 trial, 9-BW54-1 (R3 line) was tested instead of it R2 line due to seed shortage. d ‘‘n.t.’’ means ‘‘not tested’’.
575 while line 27 did not. Furthermore, R1 plants of line 49 (a medium NPR1-expresser) and line 14 (a low NPR1-expresser) also exhibited enhanced BW-resistance. For GLS, only line 49 showed enhanced resistance with a mean severity symptoms score of 1.3 vs. that of 3.9 for wild-type plants. Minor levels of increased resistance to BS were observed in three R1 lines. Lines 9 and 14 had a mean severity symptoms score of 2.8 and 2.3, respectively, vs. that of 3.3 for wild-type plants, and line 49 had a mean severity symptoms score of 1.4 vs. that of 2.0 for wild-type plants. Selected R2 lines, derived from the R1 lines exhibiting enhanced resistance to BW and FW, were evaluated for their response to the same eight diseases. The results were very similar to that observed in the parent R1 lines (Table 1), where signiﬁcant enhancement of resistance to FW and BW, moderate enhancement of the resistance to GLS and BS, and no enhanced resistance to LB, TYLCV and CMV were observed (data not shown). Since the disease severity of wild-type CL5915 plants in the ﬁrst BW evaluation was lower than the usual level (trial BW of R2 lines shown in Table 1), selected lines exhibiting enhanced BW-resistance were subjected to the evaluation again, and the results were similar to that observed in the ﬁrst screening (trial BW2 for R2 lines shown in Table 1). Some of the R2 lines were derived from individual plants that exhibited resistance to speciﬁc diseases tested in the R1 screening, and thus with the respective disease abbreviations in their line codes. R2 lines were also collected from individual R1 plants without prior selection for enhanced resistance to the diseases. No signiﬁcant diﬀerence among R2 lines collected by these two approaches was observed. However, not all the R2 lines derived from the same R1 line exhibited similar pattern of enhanced resistance. For example, only one of the 14-derived R2 lines showed signiﬁcant enhanced resistance to BW; while, surprisingly, most of the 14-derived R2 lines showed signiﬁcant enhancement of FW-resistance, which was not observed in the parent R1 line. In addition, R2 transgenic lines that accumulated higher levels of NPR1 proteins apparently may exhibit better performance in terms of the degree and/or spectrum of enhanced resistance to the diseases tested. For instance, lines 9-BW54 and 26–36, which expressed high levels of NPR1 proteins,
exhibited FW-resistance at a level similar to the FW-resistant control MH-1, in addition to enhanced resistance against BW and GLS. Line 9-BW51, which also expressed NPR1 proteins at a high level, displayed not only enhanced broadrange resistance to FW, GLS and BW, but also increased resistance to BS. To elucidate a defense mechanism possibly responsible for enhanced resistance to BW and FW observed in NPR1-transgenic tomato plants, transgenic lines exhibiting high levels of enhanced resistance to BW and FW were selected for further analysis. Disease progress was monitored in selected R2 or R3 transgenic lines, in comparison with wild-type plants and resistant control varieties. For BW, R2 lines 26–36 and R3 line 9-BW54-1 exhibited higher disease incidence over time than the resistant control H7996 did, but the diﬀerence was not statistically signiﬁcant (Figure 4). In addition, H7996 had longer latent period of symptom appearance than the two transgenic lines (Figure 4). Furthermore, the colonization progress of the pathogen of FW was monitored. R2 lines 26–36 and R3 line 9-BW54-BW8 showed similar levels of colonization with the resistant control MH1 up to 10 days after inoculation (Figure 5). Although FW pathogen colonization at the collar region of transgenic plants then reached a similar level to that found in wild-type plants, pathogen colonization at the mid-stem region of transgenic plants was still signiﬁcantly lower than that in wild-type plants. Therefore, the enhanced FW resistance in transgenic lines was mostly due to the suppression of the upward movement of the pathogen. While in MH1, the resistance was associated with the suppression of both pathogen colonization and movement.
Discussion In this study, we explored the potential use of SAR-related genes for the genetic engineering of enhanced resistance/tolerance to a spectrum of diseases in tomato. Arabidopsis NPR1 was chosen as the target transgene for generation of transgenic tomato plants, as its eﬀectiveness in conferring an enhanced degree of non-speciﬁc disease-resistance has been previously demonstrated in transgenic Arabidopsis (Cao et al., 1998) and
Figure 5. Progress of pathogen colonization at collar and mid-stem regions of transgenic tomatoes expressing NPR1 after inoculation with Fusarium oxysporum f. sp. lycopersici. CL5915 is the wild-type plant. 9-BW54-BW8 and 26–36 are the R3 and R2 lines derived from CL5915, respectively, constitutively expressing the NPR1 gene. MH1 is resistant to Fusarium wilt. The degree of plant collar and mid-stem regions colonized by the fungus are shown as percentage of colonization. Mean comparisons were conducted over tested varieties within the same sampling day. Results of the mean comparison were only presented for the days when signiﬁcant diﬀerence was identiﬁed. Columns marked with the same letters were not signiﬁcantly diﬀerent according to Duncan’s multiple-range test (P ¼ 0.05).
rice plants (Chern et al., 2001). Evaluation and extension of this approach to the tomato system is of high agronomical importance, because tomato is susceptible to a broad-spectrum of diseases, particularly in subtropical and tropical areas. Several transgenic lines with normal horticultural traits and development were generated from this work, and most of them carried a single copy of the transgene. Similarly to the NPR1transgenic Arabidopsis and rice plants, despite carrying the same transgene driven by a constitutive CaMV 35S promoter, the levels of transgene expression at both the transcriptional and translational levels varied through a wide range
among diﬀerent transgenic lines, and did not correlate with transgene copy number. In addition, although the amount of the transgenic protein(s) accumulated in individual R1 lines was in general consistent with transgenic transcript level, similar correlation was not observed in cognate R2 lines deriving from individual R1 lines. Instability of the transgenic proteins may account for this phenomenon. Overexpression of NPR1 had been previously demonstrated to confer enhanced non-speciﬁc resistance to an oomycete pathogen and a bacterial pathogen in Arabidopsis (Cao et al., 1998), and resistance to bacterial blight in rice plants (Chern et al., 2001). However, the degree and spectrum of NPR1-induced enhanced diseaseresistance were not systematically evaluated in these previous studies. Since our ultimate goal is to explore the potential use of NPR1 or other useful genes for the genetic engineering of long lasting and broad-range disease-resistance in tomato, we carried out comprehensive disease screens to determine the resultant degree and spectrum of enhanced disease-resistance in the NPR1-transgenic tomato plants. CL5915 innately processes resistance to ToMV and all the transgenic lines remained resistant to ToMV (data not shown), demonstrating that introduction and expression of the NPR1 gene in tomato plants did not aﬀect plant resistance to ToMV. Several NPR1-transgenic tomato lines displayed enhanced resistance to multiple important tropical fungal and/or bacterial diseases, including vascular as well as leaf-localized diseases. The most signiﬁcant enhancement of disease-resistance exhibited in most of the transgenic lines generated in this study was against BW and FW, and moderate enhancement of resistance to GLS and BS was also observed. These results support the eﬀectiveness of NPR1-overexpression in enhancement of resistance to fungal and bacterial diseases in plants, as previously described in transgenic Arabidopsis and rice. Most importantly, although many studies as regards enhanced disease-resistance in transgenic tomato plants have been reported (Whitham et al., 1996; Brunetti et al., 1997; Stommel et al., 1998; Kaniewski et al., 1999; Tabaeizadeh et al., 1999; Robison et al., 2001; Gubba et al., 2002; Lee et al., 2002; Li & Steﬀens, 2002), the spectrum and degree of increased disease-resistance
577 observed in the transgenic tomato plants reported in these previous studies were not as signiﬁcant as that of our NPR1-transgenic lines. The only exception may be the transgenic tomato plants overexpressing a tomato single dominant disease-resistance gene, Pto, as reported by Tang et al. (1999). However, even in this study, only enhanced level of resistance to three leaf-localized diseases was demonstrated in the Pto-transgenic tomato plants, while our NPR1-transgenic lines exhibited enhanced resistance to leaf-localized as well as vascular bacterial and fungal diseases. Results from our study demonstrate the eﬀectiveness of SAR-based approaches, particularly the overexpression of NPR1 in plants, for the genetic engineering of disease-resistance, as regards the spectrum and degree of enhanced disease-resistance. Overexpression of NPR1 in Arabidopsis and rice leads to enhanced non-speciﬁc disease-resistance in a dosage-dependent fashion (Cao et al., 1998; Chern et al., 2001). Consistent with these reports, in this study, we showed that transgenic tomato lines that accumulated higher levels of NPR1 proteins apparently exhibited higher levels and a broader spectrum of enhanced resistance to the test diseases, as demonstrated in R2 lines 9FW51, 9-BW54 and 26–36. Additionally, our results indicate that stable inheritance of enhanced disease-resistance to BW and FW may require a minimal or threshold level of NPR1 expression in transgenic tomato plants, as no consistent enhancement of resistance to BW and FW was observed in line 14, a low NPR1-expresser line, at R1 generation and R2 generation. However, the expression pattern of PR genes in our transgenic tomato plants was not consistent with that previously reported. In transgenic Arabidopsis plants overexpressing NPR1, INA-induced PR gene expression was induced in an NPR1 dosagedependent manner and correlated with the resultant enhanced resistance to bacterial and fungal pathogens (Cao et al., 1998). In contrast, although expression of a few PR genes was enhanced in some of our transgenic tomato lines, the degree of induced PR gene expression did not clearly correlate with the levels of NPR1 transgene expression, nor with the spectrum and degree of resultant enhanced resistance to the test diseases we tested. Previously, it had been shown that PR gene expression could be either NPR1-
dependent or -independent (Zhang & Shapiro, 2002), and be uncoupled from bacterial and viral resistance in Arabidopsis (Clarke et al., 1998; Wong et al., 2002). Therefore, the enhanced broad-range disease-resistance observed in NPR1trangenic tomato plants may involve the activation of other defense-related genes that were not analyzed in this study. Alternatively, some of the PR genes we tested may be involved, but diﬀerentially expressed in diﬀerent tissues. Note that the transgenic tomato lines exhibited signiﬁcant enhanced resistance to BW and FW (two soilborne vascular diseases), while displayed only limited enhanced resistance to GLS and BS (two leaf-localized diseases). However, analysis of NPR1 and PR gene expression was performed only on leaves of transgenic lines. Therefore, if the latter argument sustains, the expression pattern of the involved PR genes may correlate with that of NPR1 transgene and the resultant spectrum and degree of enhanced disease-resistance in vascular tissues, particularly in roots and collars. Further analysis of spatial expression patterns of these genes and other defense-related genes would help elucidate the relationship between NPR1enhanced resistance to the diseases we tested and defense gene expression. The enhanced resistance to BW observed in R2 line 26–36 and R3 line 9-BW54-BW8 was similar to that in H7996. Previously, it has been demonstrated that resistance to BW in H7996 is related to the internal pathogen multiplication, rather than root invasion or upward movement (Wang & Lin, 2003). Our preliminary data also indicated a similar suppression of bacterial growth in R2 line 26–36 and R3 line 9-BW54-BW8 (data not shown). However, the incidence of BW in our transgenic lines was still higher than that in H7996. These results suggest that the enhanced BW resistance induced by NPR1 overexpression in transgenic tomato may have resulted from 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 involved signal transduction pathway(s) have not been elucidated at a molecular level this far, further studies are necessary to elucidate and compare the nature of BW-resistance in H7996 and that in NPR1-transgenic tomato plants.
578 In a few NPR1-transgenic lines, the degree of enhanced resistance to FW was similar to that conferred by the single dominant gene I2 in MH1. However, detailed analysis of pathogen colonization over time revealed that the enhanced FWresistance in the transgenic lines was mostly due to the suppression of the upward movement of the pathogen, whereas the resistance in MH1 was associated with suppression of both pathogen colonization and movement. These results indicate that the enhanced FW-resistance seen in the NPR1-transgenic plants may have resulted from a partial activation of the defense system(s) mediated by I2. Alternatively, overexpression of NPR1 may activate an I2-independent defense mechanism(s) and lead to the enhanced resistance in transgenic tomato lines by suppressing the upward movement of the pathogen. Because the mechanism of I2-mediated resistance to FW and the related signal transduction pathway(s) have not been elucidated at a molecular level, we currently do not have any evidence for or against either of these hypotheses. Previously, NPR1 was shown to be involved in resistance of Arabidopsis to some diseases mediated by single dominant disease-resistance genes (Cao et al., 1994; Delaney et al., 1994, 1995; McDowell et al., 2000; Liu et al., 2002), but did not play a role in diseaseresistance mediated by other single dominant disease-resistance genes (Bittner-Eddy & Beynon, 2001; Li et al., 2001; Rairdan & Delaney, 2002; Shirano et al., 2002). However, the relationship between I2 and NPR1 has not been studied in tomato this far. Further studies on this aspect are expected to expand our understanding on FWresistance in tomato. In addition to signiﬁcant enhancement of resistance to BW and FW, overexpression of NPR1 was also eﬀective on enhancement of moderate resistance to GLS and BS in high NPR1expressing lines. No information concerning resistance to GLS at molecular level in tomato is available so far and genetically engineered resistance to GLS had not been reported before. In addition, the only reported cases of enhanced resistance to BS in tomato were achieved by overexpression of the pepper disease-resistance gene Bs2 or Pto (Tai et al., 1999; Tang et al., 1999). Therefore, our transgenic lines are of high agronomical value for their disease-resistance to a broad-spectrum of important fungal and
bacterial diseases. Our present study hence opens up new possibilities for future investigations on cross-talk mechanisms involved in tomato resistance to a broad-spectrum of pathogens. Except for the original ToMV-resistance carried in the CL5915 genome, no enhancement of resistance to other tested tomato viral pathogens, such as CMV and TYLCV, was observed in the NPR1-transgenic tomato lines (data not shown). Resistance to viral pathogens was not reported or tested in previous studies on transgenic Arabidopsis and rice plants overexpressing NPR1 (Cao et al., 1998; Chern et al., 2001). However, studies have shown that, unlike resistance to fungal and bacterial pathogens, SA-induced antiviral resistance is independent of NPR1 and PR gene expression (Chivasa & Carr, 1998; Wong et al., 2002). Accumulating evidence demonstrated in tobacco and Arabidopsis further suggested that SA-induced antiviral defense pathways could be distinct from the defense pathways leading to resistance to bacterial and fungal pathogens (Murphy et al., 1999, 2001; Kachroo et al., 2000). Therefore, overexpression of NPR1 in plants may not be eﬀective in enhancing resistance to viral pathogens, as we demonstrated in this study. In conclusion, we have demonstrated that expression of a heterologous NPR1 in tomato was eﬀective in activating certain defense signal transduction pathways, leading to enhanced resistance to a broad-range of important fungal and bacterial pathogens. Furthermore, despite exhibiting enhanced resistance to multiple diseases, no obvious deleterious eﬀects on growth and development were observed in transgenic tomato plants for over four generations (R0–R3). Particularly, enhanced resistance to FW and BW in a few NPR1-transgenic tomato plants was very similar to, but not as complete as, the resistance conferred by single dominant disease-resistance genes. This important feature could potentially reduce the selection pressure for more virulent pathogens, which may overcome the resistance conferred in the transgenic lines, and thus would be particularly desirable for the genetic engineering of enhanced resistance. In addition to the signiﬁcantly enhanced resistance to FW and BW, the moderately enhanced resistance to GLS and BS in a few transgenic lines is expected to beneﬁt tomato crop production. Moreover, possible
579 further enhancement of resistance to these as well as to other important diseases may also be achieved by appropriate application of chemical inducers or fungicides (Friedrich et al., 2001). The transgenic tomato lines we have generated could thus become an important resistance resource for breeding resistance to BW and FW, as well as be utilized as a genetic background in further breeding for resistance to a broader spectrum of diseases. Similar SAR-based strategies may also aid other important crop breeding programs for broad-spectrum disease-resistance, particularly in combination with a genetic background conferring natural disease-resistance.
Acknowledgements We thank Dr. Kenrick Deen for his critical review of this manuscript and Drs. Pierre de Wit and Jan van Kan for providing the tomato PR gene clones. We also thank Hsien-Ling Liu for the generation of transgenic lines and the technical assistance of Chiou-Fen Hsu and Hsueh-Hsiang Liau for propagating successive generations of the transgenic tomato. This work was supported by the National Science and Technology Program for Agricultural Biotechnology, Taiwan, Republic of China (Grant #: 90S201912S to C.-P. C.)
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