Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae

Plant Science 140 (1999) 71 – 79

Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae Anne Lecardonnel a,d, Laura Chauvin b, Lise Jouanin c, Antony Beaujean d, Genevie`ve Pre´vost a, Brigitte Sangwan-Norreel d,* b

a Laboratoire de Biologie de Entomophages, U.P.J.V., 33 rue St Leu, F-80039 Amiens cedex 01, France Station d’Ame´lioration de la Pomme de terre et des Plantes a` Bulbes, INRA, Domaine de Ke´raı¨ber, F 29260 Ploudaniel, France c Laboratoire de Biologie Cellulaire, INRA, route de St Cyr, F-78000 Versailles, France d Laboratoire Androgene`se et Biotechnologie, U.P.J.V., 33 rue St Leu, F-80039 Amiens cedex 01, France

Received 27 July 1998; accepted 5 October 1998

Abstract The impact of OCI (Oryzacystatin I) expressing transgenic potato on Colorado potato beetle (CPB) larvae development was investigated. Transgenic potatoes, resistant to kanamycin and expressing the OCI cysteine protease inhibitor (PI), were obtained via Agrobacterium tumefaciens genetic transformation. Four independent transgenic lines were shown by molecular analysis to exhibit a high level of OCI expression. Larvae of CPB were independently reared on either transgenic or control potato leaves until the end of larval development. A significant impact of OCI transgenic potato on larval mortality was obtained, with up to 53% mortality recorded in larvae reared on transgenic leaves. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Transgenic potato; Cysteine protease inhibitor; Colorado potato beetle

1. Introduction On account of its adaptive capacities, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) has become one of the major pests of potato. Different strategies have been put forward to overcome this phytophageous insect which is resistant to many chemical insecticides [1], transgenic plants being very promising alternatives. The use of Bacillus thuringiensis (B.t) genes encoding endotoxins active against many insects, has been extensively reviewed [2], and B.t-transgenic crops are now widely commercialised in the USA and Europe, although strains of insects resistant to this toxin have been observed [3]. Many studies have * Corresponding author. Tel.: +33-03-22-827480; fax: + 33-03-22827612; e-mail: [email protected].

also emphasised the potential usefulness of protease inhibitors (PI) in the fight against crop enemies. PI are proteins produced in storage organs and seeds. Their synthesis is induced by wounding [4–6], and they are considered to contribute to the complex defence machinery of plants [6]. Characterisation of proteases in phytophageous insects has been described elsewhere [7], and the potential use of PI for improving plant defence against insects was reviewed by Ryan [4,5]. Production of transgenic plants resistant to insects by expression of PI was first reported by Hilder et al. [8]. Since this date, many plants have been transformed with different PI, as recently reviewed [9,10]. The use of the rice Oryzacystatin I (OCI) cysteine PI for plant transformation has been de-

0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 9 7 - 6

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Fig. 1. Schematic representation of the T-DNA of the pKYV%OCI vector. P70, CaMV 35S RNA promoter with a double enhancer. V%, 5% TMV leader sequence. ocI, oryzacystatin I coding sequence. TrbcS, pea rubisco terminator. P/T 35S gus, b-glucuronidase coding sequence under control of 35S RNA regulation signals. P/T nos nptII, neomycin phosphotransferase coding sequence under control of nopaline synthase regulation signals.

scribed by several authors. Transgenic tobacco plants expressing OCI were first obtained by Masoud et al. [11]. Hosoyama et al. [12], then obtained transgenic rice expressing OCI via electroporation. However, no bioassays were undertaken in these two studies. In 1995, Benchekroun et al. [13], transformed potato and demonstrated that extracts of OCI expressing potato were active against CPB midgut extracts in vitro. In vivo experiments on Chrysomela tremulae fed with OCI expressing poplars were conducted by Leple´ et al. [14], who demonstrated increased mortality on the transgenic plants. This was the first study demonstrating that OCI expression in transgenic plant conferred resistance to Coleoptera. In addition, an engineered OCI gene was shown to confer resistance to the nematode Globodera pallida in transgenic plants [15,16]. In this report, we describe the introduction of the OCI gene [17] into potato, a major crop worldwide. This gene encodes a rice cysteine PI which has been shown to be active on CPB proteases through in vitro studies [18,19]. Transgenic plants were characterised at the molecular level to select the best candidate for insect bioassays. We demonstrated for the first time that a substantial mortality can be obtained on larvae of this important potato pest reared on OCI-transgenic foliage.

2. Materials and methods

2.1. Vector construction DNA manipulations were carried out as described by Sambrook et al. [20].Construction of vector pKYV%OCI was described in detail by Leple´ et al. [14]. This plasmid contains a kanamycin resistance gene (nptII) under control of nopaline

synthase regulation signals and the OCI cDNA [17], under the control of the CaMV 35S promoter with a doubled enhancer sequence. The TMV V% leader sequence was added in the 5% position of the OCI coding sequence to enhance translation. To create pKYV%OCI/GUS, the b-glucuronidase gene containing an intron in the coding sequence [21], under the control of the CaMV 35S promoter, was cloned into the T-DNA between the OCI and the nptII genes (Fig. 1). This plasmid was introduced in the GV3101(pMP90) strain of Agrobacterium tumefaciens [22].

2.2. Transformation of potato Two varieties of potato, BF15 (BF) and Bintje (Bi), were used in this study. The shoot cultures were grown in vitro on basal P medium as described by Masson et al. [23], except that the sucrose concentration was increased to 25 g l − 1. Cultures were maintained at 18–20°C with a day length of 16 h under 80 mE m − 2 s − 1 light intensity. The stem, petioles and leaf-pieces from 3-weekold shoots were pre-incubated overnight on the regeneration medium of Roest and Bokelmann [24]. The next day, the explants were wounded again with a scalpel blade, soaked in a culture of A. tumefaciens for a few minutes, blotted dry on sterile filter paper and placed onto the same medium. After 2 days of cocultivation at 25°C, the explants were washed with sterile water, dried on filter paper and transferred onto the regeneration medium supplemented with 50 mg l − 1 kanamycin and 250 mg l − 1 cefotaxime. Every 2 weeks, the explants were transferred to fresh medium as recommended by Visser et al. [25]. After 4 weeks, the first shoots (0.5cm) could be isolated and transferred to a P selective medium (P

A. Lecardonnel et al. / Plant Science 140 (1999) 71–79

medium with 50 mg l − 1 kanamycin) for rooting. Generally, the transformed shoots rooted in about 1 week. b-glucuronidase activity was used for early selection of transgenic material [26].

2.3. Western analysis Detection of OCI protein in transgenic material was performed by Western analysis, as previously described [14]. For protein extraction, 2 g leaf material, taken at the same position on the stem from about 3-month-old plants, were ground in extraction buffer (200 mM Tris–HCl, pH 8). After centrifugation (11000× g at 4°C), the supernatant was heated for 5 min at 65°C and centrifuged again. The total soluble protein concentration of the supernatant was estimated according to the Bradford procedure [27]. A total of 3 mg of total soluble proteins were loaded on the gels for separation. Electrophoresis of proteins was performed on SDS-PAGE using 22% polyacrylamide gels. Proteins were transferred to nitrocellulose Hybond C+ membranes (Amersham). Detection of the OCI protein by ECL™ kit (Enhanced ChemiLuminescence, Amersham) was performed according to manufacturer’s instructions, using polyclonal antisera raised in rabbits against OCI, and horseradish peroxidaseconjugated anti-rabbit IgG as secondary antibody. Purified OCI protein produced in E. coli was used as a control [14].

2.4. Southern hybridisation DNA was extracted from putative transgenic and control plants according to the procedure described by Dellaporta et al. [28]. DNA was then further purified by CsCl gradient ultracentrifugation at 55000 ×g for 20 h. After digestion with XbaI (Eurogentec), DNA was electrophoresed in 0.8% TAE agarose gel (24 h at 25 V). Alkali blotting was performed on positively charged nylon membranes (Hybond N +, Amersham) and membranes were hybridised with nptII probe labelled with [a-P]32 d[CTP] using the random prime labelling kit (Pharmacia).

2.5. Insect colony Our colony of CPB (L. decemlineata) was established using insects provided by the Protection des

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Ve´ge´taux of the Nord Pas-de-Calais region (France). In the laboratory, insects were reared continuously at 25°C, under a 18 h light/6 h dark regime. Adults were reared in plastic boxes (20× 20 ×50 cm3) closed with grill, and were fed with potato foliage harvested every day from greenhouse-grown potato plants. Egg clusters were collected every day from potato leaves and placed on filter paper in plastic boxes (15×15×8 cm3). Larvae were fed with fresh potato foliage until the end of the fourth instar. They were then transferred to new boxes (19 cm diameter, 8.5 cm depth), filled with sand for pupation. New imagos, emerging about 10–15 days later, were placed with adults and older insects were regularly removed from the colony.

2.6. Feeding assays Potato plants Bi and BF were used in the following experiment. Two independent transgenic lines were tested for each variety and compared with control plants cultivated in the same conditions (for each transgenic line coming from a distinct transformation event, a set of ten clonal replicates were grown in the same conditions). Single newly hatched larvae were placed on discs of damp filter paper in Petri dishes (5.5 cm diameter). During the whole development, larvae were fed with fresh potato leaves, which were changed every 24 h, in non-limiting quantities. Foliage was always picked from plants of the same age and from the same position on the stem. Both transgenic and control plants had been potted out for about 3 months. The number of surviving larvae, their weights and instars were recorded on days 3, 6, 9 and 13 post-hatching. Each assay consisted of three replicates of ten larvae originating from the same egg cluster. For each replicate of either transformed or control plants, the percentage of larval mortality was calculated:



LM= 1−



number of living larvae ×100 10

Analysis of variance (ANOVA) was used for data treatment, with differences between treatments being considered significant at the a =95% level. All data analyses were performed using Statview (Abacus Concepts, Berkeley, CA) on a Macintosh computer.

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Fig. 2. Comparison of the expression of OCI in the different transgenic lines by Western blot analysis. For each transgenic line and control, 3 mg of total soluble protein was loaded on the gel prior to electrophoresis. 5, 10 and 15 ng of OCI purified protein were loaded with the extracts to provide a standard for comparison of the expression levels. (A) BF variety, (B) Bi variety.

3. Results and discussion

3.1. Molecular characterisation of transgenic lines 3.1.1. OCI expression The amount of OCI protein was determined on eight transgenic lines using Western blot analysis. The relative intensity of the bands (Fig. 2), differed between the transgenic lines, showing that OCI expression was different in each of the transformed lines. The amount of OCI protein, relative to total soluble protein, was estimated by comparison with the intensity of the bands of purified protein. The four lines with highest OCI expression (i.e. BF4, BF6, Bi1 and Bi3) were chosen for further analyses. In transgenic line BF6, OCI protein represented between 0.3–0.5% of the total soluble proteins, whereas in the lines BF4, Bi1 and Bi3, OCI protein accumulated to a level of 0.16– 0.3% of the total soluble proteins. 3.1.2. Southern hybridisation Southern hybridisation was performed in order to get information on the T-DNA. DNA from four putative transgenic lines and one control was extracted, digested and subjected to electrophoresis. After alkali transfer, samples were hybridised with a nptII probe. Fig. 3 shows that T-DNA was

absent from the control, whereas it was integrated in the different transgenic lines. Moreover, the number of T-DNA copies varied among the transgenic lines, line Bi1 possessed at least four copies and line Bi3 probably had two, whereas in lines BF4 and BF6, a single T-DNA insert was detected. Thus, variations in OCI expression was not related

Fig. 3. Southern analysis of DNAs extracted from control and putative transgenic plants. For each line, 10 mg of genomic DNA were digested with XbaI enzyme. The nptII gene was used as probe. Numbers on the left correspond to molecular weights (kb).

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Table 1 Effect of OCI transgenic potatoes on the mean percentage of larval mortality of CPBa,b,c Days post hatching

3

6

9

13

Control plants Transformed plants

3.3% 11.7%

8.3% 19.2%

15% 35%

20% 40.8%

a

Each experiment corresponds to 30 larvae independently reared on control or transgenic detached leaves. b Data from both varieties were pooled, and results correspond to the mean percentages obtained for the four transformed lines (BF6, BF4, Bi1 and Bi3) or control plants (BF and Bi). c ANOVA demonstrates that results were significantly different (P=6×10−4).

to number of T-DNA inserts, but more probably to T-DNA locations in the host plant genome, as already observed in many different transgenic plants.

3.2. Lar6al de6elopment 3.2.1. Lar6al mortality Single larvae were reared in Petri dishes on control or transgenic lines, and mortality was recorded in order to test the influence of PI ingestion on CPB survival. The effect of potato variety (BF vs. Bi) and OCI expression (transgenic vs. control) on CPB larval mortality was analysed by a two-way ANOVA. No significant difference was observed between the two varieties (P \ 0.05), whereas results demonstrated that mean larval mortality, observed on the four transgenic lines (BF4, BF6, Bi1 and Bi3) was significantly higher than on control ones, along the experiment (F= 12.86, P =0.0006). The results, presented in Table 1, express the mortality of larvae reared on control or transgenic plants for both varieties. At any age (3, 6, 9 and 13 days), the mean percentage of larval mortality in transgenic plants was twice, or more, than the one observed on controls. When results were compared line by line in the different varieties (Fig. 4), some variability between the different lines could be observed. For example, ANOVA demonstrated that, in the case of the BF variety, mortality was significantly (P = 0.0001) greater on line BF6 than in line BF4, where mortality was not significantly different from control. A similar larval mortality was observed in the two Bi transgenic lines (Bi1 and Bi3), expressing OCI at the same level; this mortality was significantly

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higher than mortality of larvae fed with control leaves (P=0.0001). Thus, the presence of the OCI gene in potato had a deleterious effect on larval survival in CPB, and the difference in mortality observed among the different transgenic lines could be correlated to differences in OCI expression (cf. Fig. 2). The highest mortality was recorded on the 13th day using the BF6 transgenic line, where 53.3% of the larvae eating transgenic leaves had died at the end of experiment.

3.2.2. Lar6al growth Live larvae were weighed on days 3, 6, 9, and 13. Over the time-course of the experiment, an average 400 times increase in larvae weight was observed. Fig. 5 shows the evolution of larval weight gain, expressed as the mean weight of live larvae in the different potato lines. In the BF variety, BF4 fed larvae always had higher weight gain than larvae reared on the control line, especially on days 6 and 9 where this was significant (PB0.05). In the case of Bi variety, no significant difference could be observed between larvae fed on control and transgenic plants. In an attempt to study developmental rate, we recorded percentages of different larval instars at days 3, 6, 9, and 13, for insects reared on control and OCI transgenic lines. Table 2 shows results obtained in BF variety. Larvae fed on BF4 transgenic foliage develop at the same rate than control larvae (sometime faster), whereas larvae fed on BF6 transgenic line, developed more slowly. For example, on the 9th day only 70.6% of the larvae reared on transgenic BF6 line had reached the last larval instar (L4), whereas 88% of insects reared on control (BF) and 95.8% of larvae reared on BF4 plants were already on L4. In Bi variety, development was slowed until the 6th day in larvae fed on both Bi1 and Bi3 transgenic lines (data not shown). By the 13th day larval development was almost over and most larvae had reached the prepupal stage (L4).

4. Discussion Cysteine proteases are commonly considered to be the major type of digestive proteases in the Coleoptera gut [4,5], and especially in the CPB. Many studies have been performed to evaluate the impact of cystatins on CPB growth and develop-

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Fig. 4. Evolution of CPB Larval Mortality in the different transgenic lines of potato. Each assay was performed on 30 new hatched larvae, fed on control or transgenic potato leaves for 13 days. Mortality is reported at 3, 6, 9 and 13 days as the percentage of initial number of larvae. ANOVA demonstrated that larval mortality was significantly higher (P = 0.0001) in transgenic lines BF6, Bi1 and Bi3 than in control plants.

ment, and their possible usefulness as a control agent against this phytophageous insect has been evaluated. Wolfson and Murdock [29] were the first to demonstrate that E-64 cysteine PI had deleterious effect on CPB larval growth and development. Michaud et al. [18,19] investigated the impact of different PI and demonstrated the OCI protein from rice was able to inhibit CPB proteases in vitro. This made OCI gene a good

candidate for introduction into potato by genetic transformation, though in vivo experiments remained essential. Our study demonstrates the deleterious effect of OCI expressing potatoes on CPB larval growth and development for the first time. More than 50% CPB larval death could be observed with insects continuously reared on one line of transgenic potato foliage expressing the OCI gene. Neverthe-

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less, different mortality rates were observed between the transgenic lines, reflecting variations in OCI expression, the highest larval mortality was recorded on the highest OCI expressor (line BF6). On the contrary, in BF4 transgenic line, where OCI expression was low (near 0.1% of total soluble proteins), no additional mortality was recorded, compared to control. Larval growth and development were also investigated. Differences were observed between insects reared in normal and transgenic potatoes. Surprisingly, we showed in the BF variety, that surviving larvae had higher weight gain when they were fed

Fig. 5. Larval growth of CPB fed on OCI-transgenic or control potato leaves. Each assay was performed on 30 new hatched larvae, independently reared on potato foliage for 13 days. The growth of larvae is expressed as the mean weight of surviving larvae at 3, 6, 9 and 13 days post-hatching. Differences between BF4 and control were shown significant (ANOVA, PB0.05) for days 6 and 9.

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with transformed potatoes than those fed on controls; in addition the weight gain was different between the two transgenic lines, the highest weight gain being observed on the lowest OCI expressor line. Preliminary results on comparison of proteases observed in guts of larvae fed on normal and transgenic potatoes suggested changes in proteolytic activities. Such results have been reported by several groups. Recently, Girard et al. [30], observed a weight increase in cabbage stem flea beetles (Psylliodes chrysocephala), when feeding on one OCI-expressing transgenic line of oilseed rape, with concomitant increase in global proteolytic activity of insects. DeLeo et al. [32], describe opposite effect of low and high MTI-expressor transgenic lines, in Spodoptera littoralis. These authors suggest the existence of a threshold, representing the minimum of PI expression, necessary to induce deleterious effect. Below this threshold, larvae could overcome the deleterious effect of PI ingestion by overexpressing digestive proteases. DeLeo et al. [32], also reported increased damage in low expressing lines, and suggests this is the result of either the decrease of diet quality, or the consequence of increase of gut proteolytic capacity. These results obtained with bioassays performed on transgenic plants, strongly differ from previous observations [29,33], showing a reduction in larval weights when insects were fed on leaves coated with PI. The differences between studies could be related to the amount of ingested PI, the form of PI used, and perhaps most importantly the type of bioassay used. Surviving larvae on transgenic plants seemed to adapt to the presence of the PI in their food. Such an adaptation mechanism has already been described by several authors and recently reviewed [34,35]. It was demonstrated that insects were able to induce the synthesis of proteases insensitive to the PI they encounter, or to overexpress proteases in order to maintain a sufficient level of protease activity. Different authors also demonstrated that PI could be degradated in vitro by insensitive gut proteases of insects [19,31,36]. In conclusion, our results demonstrate that even if OCI expression induced an increase of larval mortality, the protection will not be sufficient against the CPB. Moreover, the fact that at low level of OCI expression, a gain of larval weight was observed shows the need to improve this

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Table 2 Development of CPB larvae fed on OCI-transgenic potato leavesa,b Larvae instar (% of surviving larvae) at:

BF control BF4 BF6 a b

3 days

6 days

9 days

13 days

L1

L2

L1–2

L3

L3

L4

L4

10 10.8 26.3

90 89.2 73.3

0 7.6 26.1

100 92.3 73.9

12 4.1 29.4

88 95.8 70.6

100 100 100

Each assay was performed on 30 new hatched larvae, independently reared on control or transgenic foliage. Larval instar at 3, 6 and 9 days is expressed as the percentage of surviving larvae L1, L2…L4 stand for instar 1, 2…4.

strategy. This could be obtained by engineering inhibitors for improving their interaction with the digestive proteases [37], and by the use of a combination of different resistance genes in the same construct for plant transformation [38,39], as well as the prospect of development of novel resistance genes such as lectins [40].

Acknowledgements Anne Lecardonnel was the recipient of a doctoral fellowship from the French Ministe`re de l’Education Nationale de l’Enseignement Supe´rieur et de la Recherche (MENSER). This work was partly supported by a grant from the MENSER, and the Biopoˆle Ve´ge´tal. The authors are grateful to Dr C. Foyer and Dr R.S. Sangwan for critical reading of the manuscript, Professor J.C. Wissocq for his contribution to this work, and the Syndicat de Producteurs de Plants de Pommes de Terre de la Re´gion Nord (France) for providing potato plants for insect rearing, and also thank G. Jegou for technical assistance in the greenhouse.

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