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Journal of Experimental Botany, Vol. 46, No. 284, pp. 277-283, March 1995

Journal of Experimental Botany

Changes in patterns of protein synthesis during haustorial development of Striga hermonthica (Del.) Benth. seedlings Adriana Stranger1'4, Alex Murphy 15 , Joe M. Corbett2, Mike J. Dunn2, G. Paul Bolwell3 and George R. Stewart 1 ' 6 Department of Biology, University College London, Darwin Building, Gower Street, London WCTE 6BT, UK

2

Heart Science Centre, National Heart and Lung Institute, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK Department of Biochemistry, Royal Holloway University of London, Egham, Surrey TW20 OEX, UK

3

Received 26 July 1994; Accepted 11 November 1994

Abstract This study focuses upon the developmental transition of the parasitic plant Striga hermonthica from its freeliving state (germinated seedling) to its parasitic state after development of an infection organ: the haustorium. A new method has been developed that allows the production of gram quantities of germinated and haustorially-induced Striga seedlings, thereby facilitating biochemical and molecular analysis of haustorial induction. Water-soluble proteins have been extracted from germinated seeds (stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone (2,6-DMBQ) to induce haustorium (stage B). Samples were analysed by two-dimensional polyacrylamide gel electrophoresis and quantitative as well as qualitative differences could be observed. In particular a group of four highly abundant acidic proteins (molecular weight 39 kDa, pi 5.1, 5.3, 5.3, 5.6) and three other proteins (molecular weight 12 kDa, pi 6.9; 17 kDa, pi 4.4; 17 kDa, pi 4.45) were seen in stage A while at least four proteins (molecular weight 21.5 kDa, pi 6.4; 21.5 kDa, pi 6.3; 31 kDa, pi 5.1; 34 kDa, pi 6.2) were present in greater abundance in stage B. In order to compare watersoluble protein with newly synthesized protein patterns, mRNAs from the two stages of development were isolated and cell-free translation products ana4

lysed by 2-D PAGE. Two-D gels of cell-free translation products showed the appearance of six proteins in stage B (molecular weight ranging from 10 to 35 kDa) and the presence of three acidic proteins in stage A with one protein (molecular weight 40 kDa) very similar in size to the triplet of proteins in the water-soluble protein 2-D gels. Key words: Striga hermonthica (Del.) Benth., haustorium, 2-D PAGE, 2,6-DMBQ, translation in vitro. Introduction Striga hermonthica (Del.) Benth. is an obligate parasitic flowering plant which attacks a range of hosts including maize, sorghum, millet, rice, and sugar cane causing yield losses of various degrees and leading in many cases to total destruction of staple crops (Lagoke et ai, 1988). Mature Striga hermonthica plants produce copious quantities of minute seeds (0.25-0.3 mm diameter) which require a period of pre-conditioning at specific moisture and temperature levels, and a chemical stimulant exuded from the host root for their germination (Worsham, 1987). Several root exudate chemicals, including the sesquiterpenes strigol and sorgolactone, the dihydroquinone sorgoleone-358, and a cyclic compound, comprising

To whom correspondence should be addressed at: Department of Biochemistry, Royal Holloway University of London, Egham, Surrey TW20 OEX, UK. Fax: +44 1784 43 4326. 8 Present address: Department of Rant Sciences, University of Cambridge, Downing Street, Cambridge CB3 3EA, UK • Present address: Department of Botany, The University of Queensland, Brisbane, CHd 4072, Australia. Abbreviations: 2,6-DMBQ: 2,6-dimethoxy-p-benzoquinone; PDA: 1,4 di-(1 oxo-2 propanyl) piperazine; IEF: Isoelectrtc focusing; SDS-PAGE: sodium dodecyl sulphate-polyacrytamide gel electrophoresis; PPOi 2,5-diphenyloxazole; CHAPS: {3-(3-cholamidopropyl) dimethylammonio 1-propane sulphonate]}. © Oxford University Press 1995

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Materials and methods Plant materials and growth conditions

Seeds of Striga hermonthica (Del.) Benth. were collected from Wad Medani, Sudan in 1989. Seeds were surface-sterilized for 3 min with a 5% (v/v) solution of sodium hypochlorite (10-14% (w/v) available chlorine) followed by several washes with sterile water through a Millipore filter unit. Seeds were then preconditioned by incubation on two layers of Whatman No. 1 filter paper saturated with sterile distilled water in 9 cm Petri dishes at 25 mg per Petri dish. Petri dishes were wrapped in aluminium foil to create a dark environment, and placed in an incubator at 33 °C for 7 d. Petri dishes were checked daily and supplemented with more sterile distilled water if required. Germination

After the pre-conditioning period, seeds were germinated in batches of 25 mg in 5 ml of sterile distilled water inside 7 ml gas-tight vials (Pierce) by injecting 400 jtl of 1% ethylene in nitrogen (PhaseSep) and rotating the vials for 13 h at 33 °C in the dark at 1 revolution s" 1 . This method produced germination

rates of approximately 80%. When a portion of the germinated seedlings was used to induce haustoria the unused remainder of germinated seeds were left in sterile distilled water rotating under the same conditions as the haustorially-induced for subsequent control comparison with induced seedlings. Germinated seedlings were recovered from the gas-tight vials on to Whatman No. 1 filter paper using a Millipore filter unit under very gentle water suction. Seedlings were harvested after germination (13 h) and after additional 7.5 h incubation in sterile distilled water under the same conditions as the 2,6-DMBQ-induced seedlings.

Haustorial induction

Haustorial induction was carried out by washing a 25 mg batch of germinated seeds from its filter paper disc into 50 ml of 2,6-DMBQ (1 x 10" 5 M) in a 100 ml conical flask which was then incubated at 33 °C in a rotary shaker at 180 rpm, for 7.5 h in the dark. This method gave haustorial induction rates of 70%.

Microscopy

Seedlings from the two stages of development were observed using a prototype high-definition stereo light microscope (Edge Scientific). Photographs were taken with a Nikon camera using black and white film.

Protein extraction and eiectrophoresis

Water-soluble proteins were extracted from 25 mg batches of seeds by grinding with glass beads at 4 °C using 500 y\ of TE buffer: 10 mM Tris-HCl, 1 mM EDTA pH 8.0. The. resulting homogenate was centrifuged in a microfuge at 4 °C for 20 min at 14000 rpm and the supernatant stored at - 2 0 ° C . Protein concentration was determined by the Bradford dye-binding procedure (1976).

Two-dimensional polyacrylamide gel electrophoresis 2-D PAGE was performed using the ISO-DALT system (Hoefer Scientific Instruments Ltd., Newcastle-under-Lyme, Staffordshire, UK) in accordance with the manufacturer's instructions. Briefly, first-dimension, isoelectric focusing (IEF) was carried out in 210x1.5 mm rod gels containing 3.2% T, 2.6% C polyacrylamide, 9 M urea, 4% (w/v) CHAPS, and 2% (w/v) Resolyte pH 4-8 (Merck, Poole, Dorset, UK). Protein samples were mixed with at least twice their volume of lysis buffer (9 M urea, 4% (w/v) CHAPS, 1% (w/v) DTT, 2% (w/v) Resolyte pH 4-8, and 0.5% (w/v) Bromophenol blue) prior to application to the cathodic end of the IEF gels. Samples containing 80 ^g of protein were loaded. Gels were focused at 800 V for 35000 Vh. After IEF, the gel rods were extruded on to strips of parafilm and stored at — 80 °C. The seconddimension was a gel electrophoresis performed on 230 x 200 x 1.5 mm 12% SDS-PAGE gels without stacking gels. First-dimension rod gels were loaded on to the seconddimension gel slabs while still frozen and equilibrated in situ for 5 min with 1% (w/v) DTT, 2% (w/v) SDS, 0.05% Bromophenol blue, 0.125 M Tris base, pH 6.8. The seconddimension electrophoresis was carried out at 15 mA/gel overnight at 10°C. After electrophoresis, analytical gels were fixed overnight in a methanol:acetic acidrwateT solution (5:1:4, by vol.). Protein profiles were visualized by silver staining with the Daiichi 2-D Silver Staining Kit (Kock-Light NBS, Hatfield,

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a xanthine, an unsaturated C12-carboxylic acid and a dipeptide of aspartic acid and glycine, from cowpea, have been identified as promoting germination of Striga seeds (Logan and Stewart, 1992). In addition, various other compounds such as adenine-type cytokinins, the coumarins scopoletin and hydroxycoumarin, substituted urea compounds, and sodium hypochlorite are reported to trigger germination (Logan and Stewart, 1992). It has been suggested that these compounds and the naturally occurring stimulants in root exudates may act as elicitors, stimulating endogenous ethylene synthesis and that ethylene is the actual germination signal (Jackson and Parker, 1991; Logan and Stewart, 1991). In response to a second host-derived chemical signal, the germinated seed develops an haustorium which cements it to the host root, enabling penetration, and which subsequently functions as a physiological bridge between host and parasite. Recently (Chang and Lynn, 1986), a specific haustorial inducer from the host plant, sorghum, has been isolated and identified as 2,6-dimethoxy-/>-benzoquinone (2,6-DMBQ). This induces haustoria in Striga asiatica in vitro (Smith et al., 1990; Wolf and Timko, 1991) and has been used to study patterns of protein synthesis through haustorial development (Wolf and Timko, 1992). Our research has been concerned with haustorial development of Striga hermonthica independent from the host tissue and so the term 'haustorium' refers to the specialized parasitic structure outside the host tissue which is competent to attach to the host (Riopel and Baird, 1987). In the present study we have examined aspects of biochemical development in seedlings triggered to germinate with ethylene and induced to form haustoria with 2,6-DMBQ. We report here patterns of protein synthesis following germination and haustorial development.

Haustorial development of Striga seedlings

Herts, UK). Permanent records of silver-stained 2-D patterns were made by contact-printing on to X-ray duplicating film. Computerized densitometry

Silver-stained 2-D gels were digitized at 176 pm resolution using a Molecular Dynamics 300A laser densitometer (Sunnyvale, CA, USA). Protein patterns were analysed using the PDQuest software package (Protein and DNA Imageware Inc., New York, USA) as described in Garrels (1989) running on a Sun IPX workstation. RNA extraction

Isolation of mRNA from total RNA samples

Extraction of mRNA was carried out using Promega beads (PolyA Tract mRNA Isolation System IV, Promega). mRNA was extracted at room temperature following the manufacturer's instructions at a ratio of total RNA to magnetic beads suspension of 250 ng/600 y\. Yields of up to 2% of mRNA were achieved. Cell-free translation of mRNA samples

Cell-free translation of mRNA samples was performed by using a wheat germ cell-free translation kit (Promega) and [35S]methionine (Amersham). The mRNAs extracted from germinated and haustorially-induced Striga hermonthica seedlings (300 ng each) were translated for 1 h at 25 °C following the manufacturer's instructions. The optimized exogenous

potassium acetate for our samples which gave the best synthesis of translation products was found to be 60 mM. Samples of 20 ^1 of translation products were subjected to 2D PAGE. The first dimension, isoelectric focusing (IEF) was carried out using the mini-protean II 2-D cell (Biorad) according to O'Farrell (1975). Samples were equilibrated with an equal volume of loading buffer (9.5 M urea, 5% (v/v) 2-mercaptoethanol, 2% (v/v) Triton X-100, 1.6% ampholyte pH range 5-7 and 0.4% ampholyte pH range 3-10) and loaded on tube gels (6 cm length, 0.75 cm internal diameter) containing 4% (w/v) acrylamide, 0.26% PDA [1,4 di-(l oxo-2 propanyl) piperazine], 9.2 M urea, 2% (v/v) Triton X-100, and 1.6% ampholines pH range 5-7 and 0.4% ampholines pHrange3-10. Samples were run simultaneously for 10 min at 500 V then 12 h at 700 V. Tube gels were equilibrated in sample buffer (60 mM Tris-HCl pH6.8, 2% (w/v) SDS and 5% (v/v) 2-mercaptoethanol) for 10 min. Gels were then treated according to Beiss and Lazou (1990) to remove excess 2-mercaptoethanol, by washing the tube gel in sample buffer containing 50 mM iodoacetamide for 5 min. Gels were then washed with sample buffer lacking 2-mercaptoethanol and iodoacetamide twice for 3 min each time. In the second dimension SDS-equilibrated tube gels were run on 7.5% acrylamide slab gels (0.75 x 70 x 100 mm) for 35 min at 200 V. Visualization of the cell-free translation products for 2-D PAGE gels was by fluorography using DMSO: PPO solution according to Laskey and Mills (1975). Results and discussion Germination and formation ofhaustoria

A limitation encountered in the study of haustorial development is the large-scale induction of haustoria .on germinated seedlings. To overcome this problem an in vitro system has been developed and is described here for the first time. The system facilitates the study of biochemical and molecular biological events during haustorial development and circumvents the problems often associated with studies using limited quantities of material. When using orders of magnitude such as grams of germinated seedlings, orbital shaking is introduced to aerate the medium and facilitate haustorial induction. In these experimental conditions between 70% to 90% of the germinated seedlings produced haustoria. When this procedure is omitted, no haustorial development occurred. A temperature of 33 °C for Striga hermonthica seed pre-conditioning and germination proved to be optimal (Logan and Stewart, 1991) and was reconfirmed in our experiments when processing large quantities. To induce haustoria, germinated seedlings were exposed to concentrations of 2,6-DMBQ, ranging from 10 ~2 to 10~6M. The range between 10"5 to 10" 6 M was found to be effective for haustorial induction. Higher concentrations were inhibitory. This inhibition has previously been reported for Striga asiatica (Chang and Lynn, 1986; Smith et al, 1990). Striga hermonthica follows the general scheme of primary haustorial development already described for other species of parasitic plants (Musselman and Dickison,

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Striga seedlings were harvested after ethylene or 2,6-DMBQ treatment, immediately frozen in liquid nitrogen and ground to a fine powder with a pestle and mortar. RNA extraction was performed on 500 mg aliquots (original dried weight) as described by Draper and Scott (1988). The extraction buffer: [6% (w/v) 4-aminosalicylate (BDH), 1% (w/v) tri-isopropyl naphthalene sulphonate (Kodak), 6%(v/v) Tris-buffered phenol, 50 mM Tris-HCl pH 8.4, 0.1% (w/v) insoluble Polyvinyl pyrrolidone, PVP (Sigma)] was prepared with 0.1% (v/v) di-ethylpyrocarbonate, DEPC, (Sigma) treated water. To the frozen tissue 10 ml of ice-cold extraction buffer was added. The homogenate was extracted with an equal volume of Trisbuffered phenol: chloroform (1:1, v/v), transferred to acid washed, siliconized 30 ml sterile corex tubes, and centrifuged at 8000 rpm for 10 min in a Sorval GSA rotor. The aqueous phase was transferred to a clean tube and re-extracted three more times with an equal volume of Tris-buffered phenol: chloroform (1:1, v/v). The aqueous phase was precipitated overnight with sodium acetate (pH 6.0) at a final concentration of 0.2 M with the addition of 2.5 vols of ethanol at — 20 °C. The RNA was centrifuged at 8000 rpm for 20 min in a Sorval GSA rotor at 4 °C, washed with 70% ethanol, and then resuspended in DEPCtreated water (100/^1/100 mg tissue). RNA was reprecipitated with half-volume of 8 M lithium chloride, placed on ice for 30 min and centrifuged at 8000 rpm for 30 min at 4°C. The pellet containing the RNA was washed twice with 70% ethanol and finally dissolved in 200 ^1 of DEPC-treated water. RNA quantity and purity was checked spectrophotometrically. This method yielded on average 219.5 ^g total RNA g" 1 dried weight tissue for germinated seeds. The total RNA extracted from haustorially-induced seeds was consistently more than the germinated samples by a factor of 1.8. Although the RNA preparations were always highly viscous, the total RNA was always found to be undegraded when separated on 0.8% (w/v) agarose (Sigma) gel in 1 x Tris-Borate-EDTA (TBE) buffer.

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Fig. 1. Haustorial induction in Striga hermonlhica. Radical elongation proceeds as normal in seedlings incubated in water (A), but treatment with haustorial inducer 2,6-OMBQ causes a cessation ia elongation aad (welliag io the radical tip. The radial expansion of the root merutem continues and haustorial hairs appear at 5h treatment (B, C) and by 7.5 h a fully formed primary haustorium is observed (D, E). Scale bar represents 500 ^m (A, B, D)and 125nm(C, E).

Haustorial development of Striga seedlings

Patterns of protein complements following germination

Comparison of the 2-D PAGE profiles of proteins from the two stages of development showed an increase in the expression levels of a total of eleven proteins. In stage A (Fig. 2A) increased expression of seven proteins was observed comprising a charge train of four (a, b, c, d) closely migrating protein spots of MT 39.4, pi 5.1, 5.3, 5.6, and Mz 39.1, pi 5.3 and three less abundant protein spots (e, h, i) of Mr 12, pi 6.9; Mr 17, pi 4.4; MT 17, pi 4.45. Two proteins (h, i) were not visible in stage B. Increased expression of four proteins (f, g, 1, m), of Mt 21.5, pi 6.4; Mr 21.5, pi 6.3; Mr 31, pi 5.1; Mr 34, pi 6.2 was observed in stage B (Fig. 2B) while expression levels of most proteins remained unaffected. Quantitation of these proteins is shown in Table 1. Samples from germinated seedlings taken at 13 h and germinated seedlings left in water for an additional 7.5 h showed the same protein patterns indicating that there are no time-dependent protein changes over the course of the experiment as expected (results not shown). Differentia] protein accumulation during haustorial induction has also been observed in Striga asiatica (Wolf and Timko, 1992). Although the two species: Striga asiatica and hermonthica share a morphologically similar haustorium the protein profiles are significantly different.

Table 1. Protein variations between germinated (stage A) and haustorially-induced seedlings (stage B) Protein intensity levels are expressed in arbitrary units of protein density (PDU). Data were obtained by scanning the 2-D PAGE gels of water-soluble proteins shown in Fig. 2, as described in Materials and methods. Sample

Pi

M,

Stage A

Stage B

a

5.1 5.3 5.3 5.6 6.9 6.4 6.3 4.4 4.45 5.1 6.2

39.4 39.4 39.1 39.4 12 21.5 21.5 17 \1 31 34

57 398 63931 48 858 71498 14233 939 1809 2851 T888 9292 12197

9144 2168 4115 11971 162 13190 14670 —

b c

d e

f g h i 1 m

14371 66226

Patterns of protein synthesis during transition to haustorial development

Extraction of mRNA was carried out on Striga hermonthica germinated seedlings and seedlings treated with 2,6-DMBQ for 7.5 h. Cell-free translation products were analysed by 2-D PAGE and are presented in Fig. 3. These patterns show that the abundant expression of three acidic proteins (1,8 and 9 ranging from 12 to 40 kDa) is characteristic of stage A of seed development. One of these proteins is of similar molecular weight to the four major proteins seen in the 2-D PAGE profile of watersoluble proteins. Six proteins (2, 3, 4, 5, 6 and 7 from 11 to 32 kDa) appear after 7.5 h treatment with 2,6-DMBQ (stage B). These new proteins could be the result of newly transcribed mRNA or due to enhanced mRNA stability or translatability occurring during haustorial induction. Most of the synthesized proteins remained unaltered after haustorial induction.

Conclusion

This study has demonstrated that a change in protein synthesis occurs in Striga hermonthica seedlings during the development of the haustorium. Striga hermonthica produces a complete haustorium more rapidly (7.5 h) than Striga asiatica (24 h). Two-D PAGE of water-soluble proteins showed changes in the relative abundance of at least 11 polypeptides. Cell-free translation products were analysed by 2-D PAGE and showed changes in the relative abundance of nine of the translation products. Work is in progress to isolate the genes for some of these products. This work has demonstrated that it is possible to apply standard molecular biology techniques to the study of haustorial induction in Striga species.

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1975; Riopel and Baird, 1987). The morphology during haustorial induction of Striga hermonthica seeds in response to treatment with 2,6-DMBQ was followed. Figure 1 shows photographs of representative Striga seedlings at various times during the 7.5 h induction procedure. Seedlings left to incubate in water continued radicle elongation (Fig. 1A). Those in 2,6-DMBQ ceased radicle elongation within 3 h. Radial expansion of the root meristem and initial growth of haustorial hairs was observed within 5 h (Fig. IB, C). By 7.5 h the seedlings possessed the swollen and hairy appearance of a developed primary haustorium (Fig. ID, E). This is the first report on the early ontogeny of haustorial development in Striga hermonthica seeds in response to 2,6-DMBQ. These observations are comparable to those reported for Striga asiatica (Smith et al., 1990) with the difference that haustorial formation for Striga hermonthica seeds occur within 7.5 h exposure of the germinated radicle to the haustorial inducer (2,6-DMBQ) compared to the 18-24 h observed for Striga asiatica. This difference in the rate of development is reflected in the speed of infection of host roots which was reported to be faster in Striga hermonthica than in Striga asiatica when infecting both maize and sorghum (Lane et al., 1991). This in vitro system proves to be a method which allows the study of developmental changes occurring in the parasite in response to a specific host-derived signal, independently from the host.

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4

70

Da A

30-

2tS-

44

60

70

kDa B 97-

46-

m

215-

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pi 4

Fig. 2. 2-D PAGE patterns of water-soluble proteins from (A) germinated and (B) haustorially-induced Striga herniomhka seedlings. IEF was performed on pH 4.0 to 8.0 gradient. An 80 jig protein loading was used. Gels were stained with silver. Standard rainbow molecular weight markers (Amersham) are to the left. (A) Protein spots (labelled a, b, c, d) are closely migrating and highly abundant in germinated seedlings. Proteins (e, h, i) are less abundant and (h, i) are not present in haustorially induced seedlings. (B) The expression levels of four proteins (f, g, I, m) is increased in haustorially induced seedlings. Table I shows quantification of these proteins.

Haustorial development of Striga seedlings kO 97-

- B

6946-

30-

14-

Acknowledgements The authors would like to thank Dr Vernon S. Butt for critically reading the manuscript, Andy Webb (Amersham, UK), Carol E. Lindsay and John Ball (Promega, UK) for their technical advice and support, and Jane J. Nour for her generous gift of Striga seeds. Photographs were prepared by Jon Wilson (Birkbeck College Photographic Unit). The high definition Stereo Light Microscope was a prototype supplied by Edge Scientific Instrumentation Corporation, Los Angeles, USA. This research was funded by the Science and Engineering Research Council, UK. Alex Murphy was supported by a Science and Engineering Research Council research studentship.

References Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-54. Beiss A, Lazou A. 1990. Removal of artifactual bands associated with the presence of 2-mercaptoethanol in two-dimensional polyacrylamide gel electrophoresis. Analytical Biochemistry 190, 57-9. Chang M, Lynn DG. 1986. The haustorium and the chemistry of host recognition in parasitic angiosperms. Journal of Chemical Ecology 12, 561-79.

Draper J, Scott R. 1988. The lithium chloride method for extraction of total plant RNA. In: Draper J, Scott R, Armitage P, Walden R, eds. Plant genetic transformations and gene expression: A laboratory manual Oxford: Blackwell Scientific Publications, 230-1. Garrels JI. 1989. The Quest system for quantitative analysis of two-dimensional gels. Journal of Biological Chemistry 264, 5269-82. Jackson MB, Parker C. 1991. Induction of germination by strigol and strigol analogues requires ethylene action in Striga hermonthica, but not S. forbesii. Journal of Plant Physiology 138, 383-6. Lagoke STO, Parkinson V, Agunbiade RM. 1988. Parasitic weeds and control methods in Africa. In: Kim SK, ed. Combating Striga in Africa. Ibadan, Nigeria: IITA, ICRISAT, IDRC, 22^t. Lane JA, Kershaw MJ, Moore THM, Child DV, Terry PS, Bailey JA. 1991. An in vitro system to investigate the parasitism of cereals by Striga species and resistance of cowpea to Alectra vogelii. In: Ransom JK, Musselman LJ, Worsham AD, Parker C, eds. Fifth international symposium of parasitic weeds. Nairobi, Kenya: CIMMYT. Laskey RA, Mills AD. 1975. Quantitative film detection of 3 H and 14C in polyacrylamide gels by fluorography. European Journal of Biochemistry 56, 335—41. Logan DC, Stewart GR. 1991. Role of ethylene in the germination of the hemiparasite Striga hermonthica. Plant Physiology 97, 1435-8. Logan DC, Stewart GR. 1992. Germination of the seeds of parasitic angiosperms. Seed Science Research 2, 179-90. Musselman LJ, Dickison WC. 1975. The structure and development of the haustorium in parasitic Scrophulariaceae. Botanical Journal of the Linnean Society 70, 183-212. O'Farrell PH. 1975. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007-21. Riopel JL, Baird WV. 1987. Morphogenesis of the early development of primary haustoria in Striga asiatica. In: Musselman LJ, ed. Parasitic weeds in agriculture, Vol. 1. Boca Raton FL: CRC Press, 107-25. Smith CE, Dudley MW, Lynn DG. 1990. Vegetative/parasitic transition: control and plasticity in Striga development. Plant Physiology 93, 208-15. Wolf SJ, Timko MP. 1991. In vitro root culture: a novel approach to study the obligate parasite Striga asiatica (L.) Kuntze. Plant Science 73, 233-42. Wolf SJ, Timko MP. 1992. Analysis of in vitro protein synthesis and histological studies of haustorial formation in root cultures of witchweed (Striga asiatica L. Kuntze). Journal of Experimental Botany 43, 1339-48. Worsham AD. 1987. Germination of witchweed seeds. In: Musselman LJ, ed. Parasitic weeds in agriculture, Vol. 1. Boca Raton, FL: CRC Press, 45-61.

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Fig. 3. 2-D PAGE patterns of translation products in vitro. Cell-free translation products of mRNA from germinated seedlings (A) and haustorially-induced seedlings (B). The photograph shows a detail of 2-D PAGE gels of (A) and (B). Standard molecular weight markers (Sigma) are to the left. Numbered lines indicate translation products that have been induced by 2,6-DMBQ. Open circles indicate a reduction in translation products in response to 2,6-DMBQ.

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