Amer J of Potato Res (1999) 76:169-177
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Enhanced Resistance to Bacterial Infection by E r w i n i a Carotovora Subsp. A t r o s e p t i c a in Transgenic Potato Plants Expressing the Attacin or the Cecropin SB-37 Genes Patricio Arce 1, Mauricio Morenolt, M6nica Gutierrez 3, Marlene Gebauer ~, Paola Den'Orto 1, Hebert Torres 3, Ivette Acufia 3, Pauline Oliger2, Alejandro Venegas', X a v i e r J o r d a n a ~, J u l i o K a l a z i c h 3, a n d L o r e t o H o l u ig u v-'* ~Departamento de Gen~tica Molecular y Microbiologia, Fa~ultad de Ciencias Biol6gicas, Pontificia Universidad Cat61icade Chile, P.O. Box l14-D, Santiago, Chile. ~Facultad de Agronomia, Pontificia Universidad Cat61icade Chile, P.O. Box 306, Santiago, Chile. :~CentroRegional de Investigaci6n Remehue, Instituto Nacional de Investigaciones Agropecuarias (INIA),P.O. Box 24-0, Osorno, Chile. *To whom correspondence should be addressed: Departamento de Gen~tica Molecular y Microbiologla, Facultad de Ciencias Biol6gicas, Pontificia Universidad Cat61icade Chile, P.O. Box 114-D,Santiago, Chile. email,
[email protected];telephone, (56-2) 686 2895/94. tPresent address: Departamento de Gastroenterologla, Facultad de Medicina, Pontificia Universidad Cat61icade Chile, P.O. Box l14-D, Santiago.
ABSTRACT BlacMeg and soft rot diseases, caused by the bacterium E r w i n i a c a r o t o v o r a , are a m o n g the d i s e a s e s that cause important losses in culture and storage o f potato. In this paper, w e introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, p o t a t o clones were transformed either with the gene encoding the acidic attacin protein from H y a l o p h o r a cecropia, or with the gene encoding the cecropin analog peptide SB37. These clones were evaluated for soft rot and blackleg r e s i s t a n c e , after i n o c u l a t i o n with t h e bacterial strain E r w i n i a c a r o t o v o r a subsp, a t r o s e p t i c a T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15-22%) s h o w e d i n c r e a s e d r e s i s t a n c e t o bacterial i n f e c t i o n , r e v e a l e d by reduced s e v e r i t y o f blackleg or soft rot symptoms. Expression o f the transgenes was demonstrated in some o f the clones by Northern blot analysis. This is the first report indicating that expression o f the g e n e e n c o d i n g for an a t t a c i n p r o t e i n and for t h e cecropin SB-37 peptide in transgenic p o t a t o confers increased resistance to bacterial infection.
Accepted for publication December 10, 1998. ADDITIONALKEY WORDS:Antibacterial, bacterial resistance, plant transformation, blackleg, soft rot.
INTRODUCTION Genetic engineering seems to be a promising strategy to introduce resistance against pathogens in plants. Particularly, transgenic plants expressing various genes have proven to be efficient in arresting viral and fungal proliferation (Beachy, 1997; Shah, 1997). Nevertheless, resistance against bacteria has been difficult to obtain by genetic engineering (Dtiring, 1996; Shah, 1997). Although several proteins and peptides with antibacterial activity have been described in different organisms (Boman, 1995; Broekaert et at., 1995; Hultmark, 1993; Jolles and Jolles, 1984; Molina et al., 1993a; and Molina, et at., 1993b), only a lhnited number of reports have indicated that p o t a t o e s s h o w increased resistance in plants transformed with their corresponding genes (Allefs et at., 1996; Carmona et al., 1993; Dtiring et al., 1993, Jaynes et at., 1993; Molina et at., 1993a and Wu et at., 1995). Proteins and peptides with antibacterial activity, like attacins and cecropins, are found in the humoral immune response of insects (Hultmark, 1993). Attacins are a family of proteins of around 20 kD, which act on the outer merebrane of Gram-negative bacteria by altering permeability (Carlson et al., 1991 and Engstrom et al., 1984a). The bactericidal effect of attacins has been demonstrated i n vitro on Gram-negative bacteria (Hultmark, 1993). The genes encoding for the acidic and basic forms of attacins have been isolated and c h a r a c t e r i z e d f r o m H y a l o p h o r a cecropia (Engstrom et al., 1984b; Gunne et al., 1990; and Sun et al., 1991). Although the genes encoding for attacin proteins have been proposed as promising tools for introduction of bacte-
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rial resistance into plants (Destdfano-BelWan eta/., 1990), the effect of their expression in transgenic plants has not been reported. Cecropins are a family of small amphipatic peptides of about 36 amino acids, which alter the inner membrane of bacteria by forming ion channels (Christensen et al., 1988; Hultmark, 1993; Nordeen et al., 1992; Wade et al., 1990). At low concentrations (0.1-5 DM range), cecropins are able to kill and lyse Gram-positive and Gram-negative bacteria, but not eukaryotic cells (Hultmark et al., 1982; Mills and Hammerschlag, 1993). Genes encoding peptides from the cecropin family have been isolated from different insect species (Boman et al., 1985; Gudmundsson et al., 1991; Rosetto et al., 1993). Furthernmre, a couple of synthetic analogs of crecropins with potent bactericidal activity in vitro (Dest~fanoBeltran et al., 1990; Nordeen et al., 1992) have been developed. One of these analogs is SB-37, a 38 amino acidpeptide which differs from cecropin B from H. cecTvpia by having two additional amino acids at the N-terminus and one substitution at position I 1 (Dest~fano-Beltran et al., 1990). Genes encoding for natural and synthetic cecropins have been introduced in plants to increase resistance against bacterial infection (reviewed in Dtiring, 1996). Nevertheless, results from these studies are contradictory. Transgenic tobacco plants expressing the genes for either the cecropin B peptide, the synthetic SB-37 analog peptide, or a chimeric cecropin B/cecropin A peptide (Florack et al., 1995; Hightower eta/., 1994; Jaynes et al., 1993), did not show increased resistance to infection by either Ralstonia solanacearum and/or P s e u d o m o n a s s y r i n g a e pv. tabaci. Furthermore, transgenic potato plants expressing the gene for cecropin B from H. cevropia, did not show increased resistance to infection with pathogens such as E r w i n i a carotovora subsp. atroseptica or E'rwinia chrysanthemi (Allefs et al., 1995). Nevertheless, tobacco clones transformed with the gene for Shiva, a synthetic analog of the cecropin B peptide under the control of a wound-induced promoter, produced delayed symptoms, which reduced disease severity and lowered plant mortality after infection with R. solanacearam (Dest~fano-Beltran et al., 1990; Jaynes eta/., 1993). In this work, we evaluated the possibility of introducing resistance against E. carotovora in potato, via expression of genes which encode proteins with antibacterial activity. This bacterium causes blackleg disease in stem tissue, and soft rot disease in tuber tissue (Elphinstone and Perombelon, 1986). To introduce resistance, potato clones transformed either with the gene art, encoding for the acidic attacin protein from H. eecropia (Sun et al., 1991), or with the gene sb-
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37, encoding for the SB-37 peptide (Dest6fano-Beltran et al., 1990), were obtained. These clones were evaluated in the greenhouse for soft rot and blackleg resistance, after inoculation with the bacterial stram E. c. atroseptica T7. Results reported in this paper indicate that expression of att or sb-37 genes in potato (cv. Desir6e) confers increased resistance to bacterial infection.
MATERIALS AND METHODS Genetic Constructs The plasmids containing chimeric att and sb-37 genes, cloned into the HindIII site of the pUC19 vector, were supplied by the International Potato Center (CIP, Lima, Perd) (Dest~fano-Beltran et a/., 1990). The encoding sequences for attacin and SB-37 were under the control of the double CaMV 35S promoter (Kay et al., 1987), and the 3' untranslated region of the nopaline synthase gene (Bevan et al., 1983). These genes were verified by sequencing and cloned into the H i n d I I I site of the binary vector pBI121, b e t w e e n the neomycin phosphommsferase (NPT-II) marker gene, and the ~-glucuronidase (GUS) reporter gene (Clontech Laboratories, USA). The resulting binary vectors (pBIAtt and pBISB37) (Figure 1) were introduced into A g r o b a c t e r i u m tumefaciens (strain LBA4404) via electroporation (Singh et al., 1993).
Potato Transformation Plants of potato (Solanum tube~vsum cv. Desir6e) used for transformation were propagated i n vitro in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) under controlled conditions (20-22°C, 16 h light). Transformation was carried out by infection of internode sections with A. tumefaciens harboring the binary vector pBIAtt or pBISB-37. After infection, transgenic shoots were selected for resistance to kanamycin (kan) and expression of GUS activity. Selected shoots were rooted in MS medium supplemented with 50 Dg/ml kan. Independent transgenic clones were propagated i n vitro, and then transferred to soil and grown in a greenhouse (15-30°C, 16 h light). Small tubers obtained from greenhouse-grown plants were stored at 4°C for resistance testing.
Blackleg Resistance A s s a y The bacterial strain used to evaluate resistance was E. c. atroseptica T7, which was isolated from potato stems with blackleg symptoms and was selected as the most pathogenic
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ARCE et al.: BACTERIA RESISTANT POTATO
pBIAtt RB
Hindlll
Hindlll
att
npt-Ii P nos
3'nos
pBISB-37
2P35S
Hindlll
LB
gus 3'nos
P35S
3'nos
P35S
3'nos
Hindlll
npt-II
RB P nos
3'nos
2P35S
3'nos
among 19 isolates collected in the Centro Regional de InvestigaciSn Remehue, INIA (Osorno, Chile, 40°S.L.). Collected strains were identified as E r w i n i a spp. after propagation on Crystal Violet Pectate culture medium. On this medium, E r w i n i a formed iridescent, cross-hatched translucent, deep cuplike depressed colonies, which is a characteristic of the genus E r w i n i a (Schaad, 1988). The strain E. c. at~vseptica T7 was pathogenic on potatoes, did not grow at 37°C, was negative for phosphatase activity and produced acid from (x-d glucosidase and palatinose. Isolated E. c. atroseptica T7 colonies were maintained frozen at -70°C for extended periods of storage in LB medium (Smnbrook et al., 1989) with 14% glycerol. To prepare the inoculum for the assays, the bacterium was grown on solid culture medium (3 g/l meat extract, 5 g/l bactopeptone, 2.5 g/1 glucose, 18 g/1 agar) for 48 h at 26°C and then multiplied in liquid culture medium (3 g/l meat extract, 10 g/l bactopeptone, 5 g/l NaC1) for 24 h at 26°C. After centrifugation, bacteria were resuspended in sterile distilled water at the appropriate concentration for inoculation, measured by OD at 600nm. Blackleg resistance was evaluated using plant cuttings by scoring the appearance of blackleg symptoms after inoculation. In this assay, cuttings about 5 cm-long, obtained from different greenhouse-grown plants (3 repetitions of 5 replicates each per transgenic clone), were planted in 100 ml plastic pots containing 24 g of sterile perlite. Inoculation was performed by pouring in each pot 75 ml of the bacterial suspension at the appropriate concentration (5.8x104 or 5.8x106 cfu/ml), or 75 ml of distilled water as a control. A total of 26 clones carrying the att gene; 37 clones carrying the sb-37 gene; and one clone carrying the pBI121 vector, were analyzed. In each experiment, untransformed Desir~e plants were inoculated under the same conditions. After inocula-
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FIGURE 1 Genetic constructs used f o r p o t a t o transformation. Figure shows the T-DNA region of the p B I A t t a n d pBISB-37 vectors. The art or the sb-37 gene was cloned into the HindIII site o f the pBI121 binary vector, between the neomycin phosphotransferase (npt-II) s e l e c t a b l e m a r k e r g e n e and the ~-gluc u r o n i d a s e ( g u s ) reporter gene. RB and LB, right and left borders of T-DNA region; Pnos, p r o m o t e r from t h e n o p a l i n e synt h a s e gene; 3'nos, polyadenylation signal from the nopaline s y n t h a s e gene; P35S, CaMV 35S promoter, 2P35S, double CaMV 35S promoter.
tion, cuttings were covered with a plastic bag, wrapped using a rubber band (to avoid dehydration) and placed in a room with controlled temperature (22°C-23°C). Four and six days after inoculation (DAI), the cuttings were examined for necrosis in the stem and leaves and scored by a disease index (DI) using a five-point scale (1= no symptoms; 2= small necrosis in the lower part of the cutting; 3= necrosis of the stem up to the medium part of the cutting; 4= wilt and necrosis, but the cutting still survives; 5= cutting is dead). The degree of resistance for each clone was expressed by the "resistance coefficient" (RC), calculated as follows: DI value of the untransformed control plants RC= DI value of the transgenic plants In this way, the results of several experiments performed were comparable and referred to the control in each experiment. Soft
Rot Resistance A s s a y
Soft rot resistance was assayed on tubers, by evaluating the appem~nce of rot symptoms and the sprouting capability of tubers after bacterial inoculation (Diiring et al., 1993). For this purpose, minitubers obtained from greenhouse-grown plants were cut in haft and immediately inoculated with the bacterium E. c. atroseptica T7 by vacuum infiltration (106 cfu/ml), or with distilled water as a control. Inoculated tubers were planted in soil and, 15 DAI, the number of healthy tubers that sprouted was scored. Untransformed control plants, and plants transformed with the pBI-121 vector, were included in this assay.
Molecular A n a l y s i s o f the Transgenic Plants Integration of att and sO-37 genes into the genome of transgenic potato lines were detected by PCR analysis. For
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this purpose, genomic DNA was isolated from potato leaves of transgenic and wild4ype plants, using the CTAB procedure (Ausubel, 1996), modified as described (Fang et al., 1992). DNA fragments containing sequences from the art and sb-37 genes were amplified from the genomic plant DNA, using specific primers. Primers f o r the art gene were 5'GCGCTATCGGTTCCGTAG-3' and 5'-GGCTCCCAAGAGGACTrC-3', and for the sb-37 gene were 5'-CTGACGTAAG GGATGACGC-3' and 5'-TTATCCTAGCGCTI'rGGCT-3'. Expression of the att and s b - 3 7 genes in transgenic clones was detected by Northern analysis. For this purpose, total RNA was extracted from frozen potato leaves a s described (Logemann et al., 1987). Thirty lig of total RNA were resolved on formaldehyde-agarose gels and blotted onto a Hybond-N membrane (Amersham). Hybridization was performed in a buffer containing polyethylene glycol and formamide as described (Amasino, 1986), and then washed in 2xSSC, 0.5% SDS for 10 min at 50°C. DNA fragments amplified by PCR from pBIAtt and pBISB-37 vectors using the previously described primers were used as probes. The probes were labeled with ((~-32p)dCTP by random oligonucleotideprimed synthesis (Megaprime DNA labeling system, Amersham).
clones for further evaluation using higher bacterial concentration. A control clone transformed with the pBI-121 vector (clone 6111) was included in this assay. Table 1 shows the RC values obtained 4 and 6 DM with E. c. atroseptica T7 at 5.8x104 and 5.8x106 cfu/ml, and with water as a control. Statistical analysis of the results obtained from this assay indicate that 5 from the 11 clones showed RC values significantly higher than the RC values showed by the control pBI121/6111 clone (Table 1). In this assay, control samples from untransformed plants and from transgenic plants inoculated with water did not show blackleg symptoms (Table 1). The level of resistance to soft rot was also evaluated in tubers from the clones transformed with att and sb-37 genes. In this assay, the percentage of healthy tubers that sprout 15 DM with the bacterium E. c. atroseptica T7 (106 cfu/ml) was scored. The distribution of the percent of sprouting in 35 clones carrying the att gene and in 31 clones carrying the sb3 7 gene, is shown in Figure 3A and 3B respectively. Transgenic clones were grouped according to the percent of
TABLE 1.--Level o f resistance to blackleg i n 11 selected transgenic clones.
RESULTS Transformation of Potato Plants Transformation of potato plants with the genetic constructs pBIAtt and pBISB-37, containing either the att gene or the sb-37 gene, was carried out via A. tumefaciens. Sixty and 69 transgenic clones (transformed with the att and the sb-37 gene, respectively) were selected by their capacity to root in MS-kan (50 l]g/ml) medium and by expression of the g u s reporter gene. These clones were propagated i n vitro and in the greenhouse for further bacterial resistance and molecular analysis.
Resistance Analysis of Transgenic Potato Clones Resistance to blackleg was ~~sayed in plant cuttings by evaluating appearance of disease symptoms 6 DAI with the bacterium (5.8x104 cftffml). Distribution of the RC value obtained under these conditions for 26 att clones and for 37 sb-37 clones is shown in Figure 2A and 2B, respectively. According to this distribution, 4 of 26 (15.4%) att clones and 7 of 37 (18.9%) sb-37 clones showed a promissory behavior with RC values over 1.6. We selected these promissory
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Resistance coefficient' Gene
Transgenic watel¢ clone
-pBL1216 attacin attacin attacin attacin SB-37 SB-37 SB-37 SB-37 SB-37 SB-37 SB-37
w t~ 6111 84 102 15 104 31 15 22 99 42 21 311
5.8x1~ cfu/ml3
6DM 4
4DM
1.00±0.00 1.00_+0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00+0.00 1.00±0.00
1.02±0.02 1.18±0.39 3.28±0.90* 3.41±1.03" 2.36±1.02" 2.56±1.26 3.41±1.03" 3.03±0.90* 2.30±1.19 2.21±1.23 2.67±1.31 2.02±0.74 1.73±0.78
6DAI
5.8x10 ~cfu/nff3 4DM
6DAI
1.02±0.02 1.12±0.22 1.01±0.02 1.21±0.56 1.28±0.49 1.07±0.22 2.96±0.97* 2.08±0.42* 2.04±0.51" 2.88±1.06" 2.83+L03" 2.15±1.07" 1.93±0.48 2.31±1.59" 1.59±0.74 2.28±1.56 1.63±0.24 1.49±0.19 3.47+1.04" 1.68±0.29" 1.68+0.25" 2.59±1.34 1.74±0.19" 1.53±0.25 2.39±1.29 1.86±0.81 0.81±0.85 2.69±1.60 1.50±0.70 1.51±0.81 2.06±0.85 0.95±0.23 1.17±0.22 2.34±1.21 1.66±0.53 1.94±0.57 1.75±0.74 1.51±0.34 1.43±0.26
1Resistance coefficient values are man ± SD of 3 repetitions (5 replicates each). 2Control samples inoculated with water. 3Bacterial concentration used for inoculation. 4Days after inoculation. 5Resistance coefficient values for control untransformed plants are mean + SD of 7 experiments (3 repetitions of 5 replicates each). DI values obtained for control untransformed plants (mean ± SD of 7 experiments) are 3.74 ± 0.45 (5.8x104 cfu/m], 4DAI), 4.25 ± 0.42 (5.8x104 cfu/ml, 6DAI), 4.13 ± 0.60 (5.8x106 cfu/ml, 4DAI) and 4.57 ± 0.30 (5.8x106 cfu/ml, 6DM). 6Control clone transformed with the pBI121 vector. *RC values significantlydifferent from the control (P
