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Molecular Breeding 13: 49–57, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Transgenic Nicotiana Tabacum L. with enhanced trichome exudate cembratrieneols has reduced aphid infestation in the field
Erming Wang, James T. Hall and George J. Wagner*
Plant Physiology/Biochemistry/Molecular Biology Program, Agronomy Department, 200L KTRDC, University of Kentucky, Lexington, KY 40546-0236, U.S.A.; *Author for correspondence (e-mail: gwagner@uky.edu; phone: 001-859-257-5974; fax: 001-859-323-1077)
Received 8 January 2003; accepted in revised form 20 May 2003

Key words: Aphid resistance, Cembratrieneols, Natural-product-based resistance, Nicotiana Tabacum L., P450, Trichome. Abstract Glandular trichomes of many plants secrete natural products that influence plant/insect interactions. For example, tobacco varieties having relatively high cembratrieneols ͑CBTols͒ are known to have enhanced aphid resistance in the field. CBTols and corresponding CBTdiols comprise ~1.4 and ~62%, respectively, of trichome exudate in the aphid susceptible tobacco variety, T.I. 1068. Using this cultivar we suppressed the CYP71D16 gene that encodes the enzyme that converts CBTols to CBTdiols. In suppressed plants CBTols and CBTdiols accounted for about 27 and 35% of exudate weight, respectively. Total CBTols plus CBTdiols was not changed substantially. Here we studied the relationship between aphid infestation and increased exudate CBTols in the field using self progeny derived from 5 independent primary transgenic T. I. 1068 plants having suppressed CYP71D16 activity. Two hundred individual plants were scored for aphid infestation, and their trichome exudate compositions were determined by gas chromatography. A significant negative correlation was found between high CBTols levels in trichome exudates and aphid infestation. No aphid infestation was observed on the majority of plants with CBTols/CBTdiols ratios of Ͼ 1.49, which represents a Ͼ 20-fold increase in CBTols, and a Ͼ 40% decrease in CBTdiols over control T.I. 1068 exudate. In contrast, aphid infestation occurred on most plants with CBTols/ CBTdiols ratios Ͻ 0.201, which is similar to that of the untransformed control T.I. 1068. These results demonstrate the feasibility of using metabolic engineering of glandular trichomes to enhance natural product-based pest resistance. Introduction Most flowering plants bear trichomes on their aerial surfaces. Trichomes may be non-glandular, nonsecreting, or glandular-secreting, depending on the species. An apparent function of exudates ͑secretions͒ of glandular-secreting trichomes is to confer resistance to insects through toxicity, physical entrapment, or alteration of feeding behavior ͑Wagner 1991͒. Depending on the species, trichome exudates can accumulate to a level up to 30% of leaf dry weight ͑Fahn 1988͒. In well studied systems, it is established that specific exudate constituents effect insect resistance ͑e.g., Duffey 1986; Jackson and Danehower 1996; Schultz et al. 1996͒. Removal of the glandular trichomes or the exudates of tomato by rinsing foliage with solvents causes reduced resistance to insects ͑Hawthorne et al. 1992͒. Terpenoids are the most abundant trichome exudate constituents, but phenolics, quinones and flavonoids are also prominent in certain plant species ͑Kelsey et al. 1984͒. Aphids are a major pest of Nicotiana tabacum L. in most of the tobacco-growing countries of the world, and development of aphid-resistant tobacco

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Figure 1. Gas chromatographic analyses of the trichome exudates from T1 plants. A: a control-like plant ͑plant FC35T1-33͒, B: a plant with moderately suppressed CYP71D16 activity ͑plant FC35T1-38͒, C: a plant with highly suppressed CYP71D16 activity ͑plant FA3T1-36͒. Typical profiles shown were quantified by inclusion of a known amount of 1-tricosonal as an internal standard. Relative detector responses of CBTols and CBTdiols in Figure 3B, Figure 3C were compared to Figure 3A after adjustment using the internal standard response. This comparison showed that the sum of corrected detector responses for all CBTs was similar in the 3 cases.

cultivars using conventional breeding and metabolic engineering is an alternative to the use of synthetic chemical aphicides ͑Johnson et al. 2002; Wagner and Wang 2001͒. Early studies indicated that trichome exudates are key components in tobacco/insect interactions ͑Severson et al. 1985; Jackson and Danehower 1996; Severson et al. 1994͒. Principal trichome exudate components of tobaccos are the cembranoid diterpenes ␣- and ␤-cembratrienediols ͑CBTdiols͒, the precursors of these ͑the ␣- and ␤-CBTols͒, labdanoid diterpenes ͑cis-abienol and labdenediol͒, and sugar esters ͑Severson et al. 1985; Johnson et al. 2002͒. In the tobacco cultivar used in this study ͑T.I. 1068͒, CBTdiols are the main components, account-

ing for ~62% of the total exudate weight and ~10% of leaf dry weight. In contrast, the CBTols ͑precursors of the CBTdiols͒, constitute only ~1.4% of the total exudate weight ͑Wang and Wagner 2003͒. Studies on the influence of trichome exudate components on aphid infestation have demonstrated a clear relationship between aphid resistance in tobacco and differences in the composition of trichome exudates ͑Severson et al. 1994; Johnson et al. 2002͒. CBTols, cis-abienol and suger esters are toxic to tobacco aphids ͑Severson et al. 1994͒ when applied directly to insects. Relatively high CBTols producing tobaccos show decreased aphid infestation in the field ͑Jackson and Danehower 1996͒.

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Table 1. The CBTols to CBTdiols ratios and aphid resistance ratings of individual plants from different segregating populations within different replications Plant number Replication I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 FA3T1 0.09/1 0.08/4 0.09/1 0.08/1 1.88/0 3.21/0 1.89/0 4.17/0 0.11/2 1.37/0 4.22/0 2.20/0 0.05/2 2.35/1 1.51/0 4.24/0 0.07/3 0.04/2 0.21/3 0.04/0 0.06/0 1.96/0 3.19/0 1.97/0 0.08/1 0.06/2 0.10/4 0.06/2 2.90/0 0.06/2 0.06/2 0.06/1 0.08/3 0.06/4 0.08/2 4.09/0 4.41/1 0.08/4 0.07/3 0.04/1 FA16T1 1.23/0 0.06/2 0.87/0 0.17/4 4.73/0 1.37/0 3.53/0 0.07/1 3.61/0 3.95/0 0.08/0 0.64/0 1.90/0 0.07/4 2.20/0 1.97/0 0.94/0 2.87/0 2.23/0 0.07/1 2.48/0 2.76/2 2.02/0 7.50/0 0.06/1 2.58/0 0.32/2 1.80/0 2.11/0 1.70/0 1.61/0 2.72/0 1.27/1 1.49/0 1.81/0 1.95/1 0.10/3 3.03/1 0.07/1 0.73/0 FA17T1 0.12/0 1.24/0 2.57/0 6.10/0 0.07/1 0.46/0 0.09/1 2.05/0 0.09/1 0.05/0 0.07/0 1.83/0 3.05/0 4.87/0 0.08/2 1.08/2 0.09/1 0.07/0 0.15/0 2.69/0 1.68/0 2.24/0 2.82/0 0.10/0 0.10/1 1.29/0 0.07/3 0.09/1 2.51/0 2.49/0 4.15/0 2.56/0 1.98/0 7.51/0 2.34/1 2.29/0 0.09/2 2.76/0 2.23/1 1.72/0 FC35T1 0.63/1 0.63/2 1.49/0 0.17/1 2.84/0 1.71/0 0.55/1 0.12/0 1.83/0 0.68/0 0.41/0 0.56/3 1.09/0 0.14/1 2.32/0 1.58/0 0.10/0 1.49/1 0.07/1 6.28/0 0.15/0 0.07/2 0.11/1 0.26/0 0.54/2 0.06/4 3.25/0 2.44/0 2.24/0 1.99/0 2.35/0 0.44/2 0.09/2 0.27/1 0.20/1 1.76/1 0.11/2 1.17/0 1.21/1 2.06/0 QS2 0.15/2 0.08/2 0.06/2 0.09/1 0.09/1 0.15/4 0.09/2 0.08/2 0.09/4 0.12/2 0.18/2 0.08/4 0.09/4 0.05/1 0.09/4 0.08/4 0.08/2 0.09/1 0.09/1 0.07/3 0.12/2 0.06/2 0.07/1 0.10/2 0.09/2 0.07/4 0.09/2 0.08/4 0.04/2 0.06/2 0.05/2 0.05/1 0.06/2 0.03/2 0.07/2 0.12/4 0.08/4 0.10/4 0.11/3 0.04/2

Replication II

Replication III

Lower case numbers are the CBTols to CBTdiols ratios for individual plants, and the numbers in italics are aphid resistance rating.

Conversion of CBTols to CBTdiols is catalyzed by a cytochrome P450 hydroxylase encoded by a gene expressed in tobacco glandular trichomes. We isolated this gene ͑Wang et al. 2001͒, designated as CYP71D16 ͑Nelson͒. Suppression of CYP71D16, either by antisense inhibition or sense co-suppression,

caused a dramatic decrease in CBTdiols and a substantial increase in CBTols in primary transformant tobaccos ͑Wang et al. 2001; Wang and Wagner 2002͒. The total CBT level ͑CBTols plus CBTdiols͒ was not changed substantially. Exudates of transgenic plants with increased CBTols showed higher aphidicidal ac-

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Table 2. Variation of CBTols to CBTdiols ratios and aphid infestation ratings for the progenies analyzed Progenies CBTols/CBTdiols ratios Aphid infestation ratings FA3 T1 1.184 Ϯ 1.533 1.3 Ϯ 1.36 FA16 T1 1.767 Ϯ 1.534 0.6 Ϯ 1.08 FA17 T1 1.696 Ϯ 1.754 0.4 Ϯ 0.75 FC35 T1 1.137 Ϯ 1.237 0.8 Ϯ 0.98 QS2 0.084 Ϯ 0.031 2.4 Ϯ 1.08

Numbers in bold are averages for plants sampled ͑40͒ of each progeny, while numbers in italics are the standard deviation.

Figure 2. PCR analysis of T1-plants derived from the primary transgenic plant FC35 expressing the full-length CYP71D16 coding region in antisense orientation. Lanes 1-3, PCR amplification products using the primers EW 79 and GSP-2; Lanes 4-6, PCR amplification products using the primers EW 82 and GSP-1; Lanes 7-9, PCR amplification products using the primers GSP-3 and GSP-4. DNA templates for lanes 1,4,7, were from plant FC35 T1-31 ͑a HS plant͒; DNA templates for lanes 2,5,8 were from plant FC35 T1-32 ͑a MS plant͒; DNA templates for lanes 3,6,9 were from control T.I.1068.

tivity in the laboratory, and these plants showed greatly diminished aphid colonization responses in the greenhouse ͑Wang et al. 2001͒. A limited number of transgenic lines were used in these greenhouse studies. To expand these studies we have evaluated the relationship between CBTols to CBTdiols ratios of trichome exudates and aphid infestation of tobacco plants growing in the field using self progenies derived from the primary transgenic plants with CYP71D16-suppressed exudate profiles. CBTols to CBTdiols ratios were determined for each of the 200 plants tested, and were correlated with aphid infestation ratings of the same individual plants. Samples were taken for exudate analysis and aphid ratings were made at the same time. Generally, plants with high CBTols had lower aphid infestation. Our results support those of earlier greenhouse tests and represent

a further step towards the application of metabolic engineering of natural product-based pest resistance. Materials and methods Plant materials, field design The field experiment was carried out at the University of Kentucky Agricultural Experiment Station, University of Kentucky, Lexington, Kentucky during the summer and early autumn of 2001. Plant materials used were T1-derived progenies from the primary transgenic plants FA3, FA16, FA17, FC11, and FC35, and the T2-derived progeny from the primary transgenic plant 1-31-2Q. About 150 seeds from each line were grown to a 6-leaf stage then transplanted onto a floating tray filled with promix soil over a water bed

53 PCR analysis The presence of the transgene in progeny was verified by polymerase chain reaction ͑PCR͒. The primer sets used were: i͒ a primer ͑EW82͒ within the 35S promoter region combined with a primer specific to the transgene ͑CYP71D16͒; ii͒ a primer ͑EW79͒ within the rbcS terminator region combined with a transgene specific primer. The sequences for primers EW79 and EW82 were 5’-GAACTTGACGAACGTTGTCGAAAC-3’and 5’-CACGCTGAAATCACCAGTCTC-3’, respectively. The sequences for the 2 gene specific primers, GSP-1 and GSP-2, were 5’-GGCACTGAGCAATTCCAAGAGACA-3’ and, 5’-CATCTTTGGTGGGGGAACAGA-3’, respectively. In addition, a primer set specific to CYP71D16 gene was used to ensure that the DNA template was suitable for PCR amplification when no PCR amplification of the transgene occurred. The sequences for this primer set were: 5’-TTCAGACTGCATCCTCCACTACC-3’ ͑forward primer͒ and 5’-TCCAGTCAAAGTGATTCAAC-3’ ͑reverse primer͒. Tobacco genomic DNA was isolated using the DNeasyTM Plant Mini Kit ͑QIAGEN Inc., Valencia, CA͒. Taq DNA polymerase along with its buffer was purchased from InvitrogenTM, Carlsbad, CA. The cycling conditions were: 50 ng genomic DNA for each PCR reaction; initial denaturation at 94 °C for 4 min; 36 amplification cycles, each consisting of denaturation at 94 °C for 30 second, annealing at 56 °C for 30 seconds, and extension at 72 °C for 1 min 30 seconds; and post-extension at 72 °C for 5 min. Analysis of leaf surface chemistry Leaf surface exudates of individual plants were collected according to Wang et al ͑2001͒. Briefly, 2 leaf discs ͑each 2 cm in diameter͒ were sampled from individual plants, and each was dipped 6 times for 5 seconds each in 10 ml of methylene chloride in a 20-ml scintillation vial. Samples were analyzed immediately by gas chromatography ͑GC͒ or were stored in a freezer at Ϫ 80 °C until analysis. GC analysis was carried out as previously described ͑Wang et al. 2001͒. Earlier research indicated that total CBTs ͑CBTols plus CBTdiols͒ of primary transgenic plants was not changed from that of control, non-transgenic plants ͑Wang et al. 2001͒. Therefore, the ratios of CBTols to CBTdiols ͑termed CBT ratio͒ of exudates from individual plants were used in this study to represent the CBTols levels of individual

Figure 3. Aphid infestation response on T1-plants with different levels of CYP71D16 suppression. A, plant FA3 T1-38 ͑controllike͒; B, plant FA3 T1-36 ͑an HS plant͒. Arrow points to an alate aphid.

in the greenhouse ͑32 °C, natural light͒. On 10 July, the seedlings, about 5-inch in height, were transplanted into the field. Plots were arranged in a randomized complete block design with 3 replications. Each plot consisted of rows, 3.5 feet wide, 42-inches apart and 54 feet long. Each row contained 32 individual plants, spaced at 20 inch apart. The space between blocks was 5 feet. Conventional cultural practices for growing tobacco were followed during the course of this experiment, except that no insecticides were applied to either the tobacco plants or the soil. Weeds were removed manually.

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Table 3. Distribution of aphid infestation versus CYP71D16-suppressed phenotype Progenies Aphid infestation ratings 0 FA3 T1 HS 13 MS 1 LS 2 HS 19 MS 7 LS 1 HS 19 MS 3 LS 6 HS 12 MS 6 LS 3 HS 0 MS 0 LS 0 HS 63 MS 17 LS 12 1 2 0 6 2 1 4 2 0 6 1 5 5 0 0 7 7 6 28 2 0 0 8 1 1 1 0 1 2 0 3 3 0 0 20 1 5 34 3 0 0 4 0 0 1 0 0 1 0 1 0 0 0 1 0 1 7 4 0 0 4 40 FA16 T1 0 0 2 40 FA17 T1 0 0 0 40 FA35 T1 0 0 1 40 QS2 0 0 12 40 All progenies pooled 0 0 19 200 HS, stands for high level of suppression ͑CBT ratio Ͼ 1.49͒; MS, moderate level of suppression ͑CBT ratio between 0.201 and 1.49͒; LS, low level of suppression ͑CBT ratio Ͻ 0.201͒ Total number of plants for each progeny

Monitoring of Aphid Infestation The aphid species naturally infesting tobacco plants in this study was the red aphid Myzus nicotiana, Blackman. At the flowering stage ͑late September, 2001͒, individual plants from each line were scored for aphid infestation on a scale of 0 to 4: 0, no aphid infestation; 1, light infestation; 2, moderate infestation; 3, heavy infestation; 4, very heavy infestation. Scoring was followed by collection of exudates ͑above͒. A visual image was used for scoring in which about 12, 9, 6, 3, and 0 apterous aphids per about 1.5 cm2 on young leaves represented very heavy, heavy, moderate, light, and no infestation, respectively. Statistical analysis Correlation coefficients between aphid infestation rating and the CBT ratio were calculated using the insert function in Microsoft Excel XP ͑2001 version͒.

Figure 4. Frequency distribution of aphid infestation. HS, high level of suppressed CYP71D16 activity; MS, moderate level of suppressed CYP71D16 activity; LS, low level of suppressed CYP71D16 activity ͑control-like͒. Aphid infestation ratings: 0, none; 1, light; 2, moderate; 3, heavy; 4, very heavy.

plants. This measure is independent of sampling variations that may occur in the GC analysis, and directly reflects the trait being studied.

55 Results and discussions Production of transgenic progenies Three independent primary transgenic plants expressing the CYP71D16 gene sequence in sense orientation ͑co-suppression, FA3, FA16, FA17͒ and two independent primary transgenic plants expressing this gene in antisense orientation ͑FC35, FC11͒ were used to produce T1 progenies for this study. All 5 parent plants showed high levels of gene suppression ͑GC profiles similar to that shown in Figure 1C͒. The T1 progeny were named FA3T1, FA16T1, FA17T1, FC11T1 and FC35T1, respectively. In addition, T2 progeny derived from the primary transgenic plant 1-31-2Q that expressed part of CYP71D16 gene sequence in antisense orientation ͑Wang et al. 2001͒ were tested. Both the primary transgenic plant 1-31-2Q and its T1 plant, from which QS2 was obtained, had a moderate level of suppression of CYP71D16 gene activity, with a GC profile similar to that shown in Figure 1B. In our previous study, Plants FA3 and 1-31-2Q were tested for aphid resistance in greenhouse experiments, and both plants showed a high level of resistance to aphid colonization ͑Wang et al. 2001͒. No greenhouse experiments were performed to evaluate aphid resistance in the other 4 primary transgenic plants ͑FA16, FA17, FC11 and FC35͒. In the field, all lines except for FC11T1 had control-like growth ͓like transgenic plants having unaltered CBT ratios ͑Figure 1A͒, and untransformed plants in a nearby field͔ and were similar in size. Poor germination of seeds occurred in FC11T1, and delayed growth was observed in some of the plants. Since delayed growth might effect colonization by aphids, FC11T1 progeny were not studied further. GC and PCR analyses For each plot, starting from the first plant next to the space between replications, 10 ͑for replication I͒ or 15 ͑for replications II and III͒ consecutive individual T1 ͑or T2 for QS2͒ plants were selected for sampling, and the exudates from each individual plant were subjected to GC analysis. The GC profiles obtained for all progeny can be classified into 3 levels: low level, or control-like suppression ͑termed LS͒, a moderate level of suppression ͑termed MS͒, and a high level of suppression ͑termed HS͒. CBTdiols are the major component in exudates of LS plants, while CBTols are only present in trace amounts ͑Figure 1A͒. In contrast, in HS plants, the CBTdiols are reduced to a trace level due to the suppression of the CYP71D16 gene, and their direct precursors, the CBTols, are dramatically increased ͑Figure 1C͒. MS plants had CBTols levels well above that of LS plants, but much lower than those of HS plants ͑Figure 1B͒. As shown in Figure 1, the amount of cis-abienol was relatively unchanged in transgenic plants. This was also the case for sugar esters that elute after 42 min ͑not shown͒. This was expected because synthesis of these compounds is independent of the CYP71D16 gene. The presence of the transgene in T1 or T2 ͑QS2͒ progenies with highly or moderately suppressed GC profiles was verified using PCR. Figure 2 shows PCR amplification products derived from FC35T1 progeny. Plant FC35 contained transgenic copies of the CYP71D16 gene in antisense orientation. Specific PCR amplification by the 2 primer sets ͑EW82 and GSP-1, and EW79 and GSP-2͒ should occur in FC35T1 plants that possess the transgene. As Figure 2 reveals, an expected ~ 600 bp PCR product amplified by the primers EW79 and GSP-2 was detected in a HS T1 plant ͑Lane 1͒ and a MS T1 plant and ͑Lane 2͒, whereas no amplification occurred in the untransformed control ͑Lane 3͒. Similar results were observed when the primers EW82 and GSP-1 were used ͑Figure 2, lane 4, 5 and 6͒. An expected ~250 bp fragment, amplified by the CYP71D16 specific primers GSP-3 and GSP-4, appeared in all 3 plants ͑Figure 2, Lane 7, 8, and 9͒. This rules out the possibility that the lack of PCR amplification in the control case using the primer sets ͑EW79 and GSP-2, and EW82 and GSP-1; Figure 2, lane 3 and 6, respectively͒ was due to a poor quality genomic DNA in the control case. In order to quantitatively describe the level of CYP71D16 suppression, the CBT ratio was calculated from GC profiles of all individual plants. The CBT ratio for untransformed T.I. 1068 is usually between 0.03 and 0.05, and rarely above 0.10 ͑Wang et al. 2002; E Wang and GJ Wagner, unpublished data͒. The CBT ratios for each individual plant in all progenies are listed in Table 1, and Table 2 displays the average CBT ratio for each progeny. As shown in Table 1, Table 2, the average CBT ratio for QS2 was the lowest among the 5 progenies tested, and little variation was found in the CBT ratios among individual plants of this progeny. All QS2 progeny ͑from an antisense parent͒ had GC profiles similar to

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Table 4. Correlation between CBTols/CBTdiols ratios and aphid infestation ratings Progenies FA3 T1 FA16 T1 FA17 T1 FC35T1 QS2 All progenies pooled Number of plants observed 40 40 40 40 40 200 Correlation coefficient ͑r͒ Ϫ 0.62 Ϫ 0.44 Ϫ 0.41 Ϫ 0.46 0.19 Ϫ 0.50 P values Ͻ 0.01** Ͻ 0.01** Ͻ 0.01** Ͻ 0.01** Ͼ 0.05 Ͻ 0.01**

** represents significance at the1% level.

that of the untransformed control T.I.1068. This result might be caused by the unstable transmission of gene silencing in this antisense progeny through meiosis, due to its epigenetic nature ͑Bruening 1998͒. Considerable variation in CBT ratios was observed among plants in the other 4 progenies tested ͑Table 1, Table 2͒. We speculate that the large variation in CBT ratios in the 4 sampled populations was due to differences in silencing efficiency and in transgene composition among T1 individuals. Aphid Infestation All individual plants analyzed by GC were scored visually for aphid infestation based on a scale of 0 to 4, with 0 being essentially no aphid infestation ͑Figure 3B͒ and 4 the heaviest infestation ͑Figure 3A͒. As Table 1, Table 2 indicate, all of the QS2 plants incurred aphid infestation, and the majority had at least a moderate infestation. The other 4 lines showed a wide range of variation in aphid infestation, from no infestation to the heaviest observed, most likely due to large differences in CBT ratios observed among individual T1 plants. The interaction of aphids and exudate is a complex process that is influenced by exudate composition, alate aphid migration, and undoubtedly other environmental factors. In earlier studies ͑Wang et al. 2001͒ alate aphids were observed on CYP71D16 suppressed as well as control plants, suggesting that alate aphid migration was not reduced by gene suppression while colonization was. Similarly, alates were observed on highly suppressed field plants that lacked colonization with apterous aphids ͑Figure 3, arrow͒. Frequency Distribution for Aphid Infestation Exudates of all HS plants had CBT ratios of Ͼ 1.49, which represents a Ͼ 20-fold increase of CBTols and

a Ͼ 40% decrease of CBTdiols, relative to that of the unsuppressed control. In contrast, LS plants had a CBT ratio of Ͻ 0.201, reflecting a trichome exudate composition like that of control. The CBT ratios of the MS plants were less than or equal to 1.49 but greater than or equal to 0.201. For each of the suppression levels ͑HS, MS and LS͒ the frequency distribution for aphid infestation was investigated in order to assess the relationship between aphid infestation and higher CBTols content. The frequency distribution for aphid infestation for progeny of each line is detailed in Table 3, and the pooled data are illustrated in Figure 4. As shown in Figure 4, no aphid infestation was found in about 90% of HS plants, and none of the HS plants had higher aphid infestation rating more than 2 ͑moderate͒. In contrast, aphid infestation occurred in about 90% of the LS plants, and nearly 20% of LS plants incurred the heaviest aphid infestation ͑Figure 4͒. No aphid infestation was observed on ~60% of the MS plants whereas the rest were colonized ͑Figure 4͒. Correlation Analysis Significant negative correlations between aphid infestation ratings and CBT ratios were observed for all progenies except QS2 ͑Table 4͒. The lack of correlation between aphid infestation rating and CBT ratios for the progeny QS2 is due to the low variation of CBT ratios among the individual plants ͑Table 1, Table 2͒. Correlation analysis of the pooled data ͑all progenies included, 200 plants͒ also indicated a significant negative correlation between aphid infestation ratings and CBT ratios ͑Table 4͒. In summary, results of this field study substantiate results obtained in earlier greenhouse studies ͑Wang et al 2001͒ and support the role of CBTols in providing natural-product-based aphid resistance in tobaccos. Results also show the usefulness of metabolic

57 engineering for increasing endogenous insect resistance mechanisms in plants. Acknoledgements We thank Dr. R. Miller for assistence with field work and we thank Dr. Dennis Egli for reviewing this manuscript. The work was supported by a grant to GJW from the Kentucky Tobacco Research and Development Center. References
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