accum ulation during haustorial induction, water soluble proteins were extracted at different stages of development (see section 2.4.1a). Samples of S. hermonthica seed were germinated and haustorially induced as described in Chapter 3, but in 50 mg batches.
To exam ine any differential accum ulation, protein sam ples extracted from germinated seedlings at 1.5 h, 3 h, 5 h and 7.5 h after treatment with 2,6-DMBQ and were subjected to SDS-PAGE analysis. The results are shown in figure 5.1.
7
3 1 . 5 water 2 0 0 1 1 6 9 7 .4 6 6 4 5 31 21.5 r- Fi g 5.1Water soluble polypeptide composition during haustorial induction. Lanes 2-5 contain protein extracted after 1,3,5 and 7.5 h treatment with 2,6-DMBQ, lane 1 contains protein from control seedlings incubated in water for 7.5 h. Markers (kD) are along the left side.
The number of polypeptides present in each lane made interpretation of the gel difficult. However, one peptide with an
approxim ate molecular weight of 29 decreased within 1.5 h o f treatm ent with 2,6-DMBQ and two peptides with molecular weights of 100 and 40 increased at this time. One dimensional SDS-PAGE was not sensitive enough to reveal detailed changes. 1-D SDS-PAGE separates polypeptides on the basis of their mass alone, thus each band on the SDS-PAGE gel may contain many different species of polypeptides that m erely share the same mass. Hence a decrease or increase in a minor polypeptide com ponent can be easily masked. As a result of this investigation, it was decided to increase the resolution of the protein profiles by using 2-D e le c tro p h o re s is .
Two dimensional electrophoresis (lEF/SDS-PAGE) was carried out on w ater soluble proteins extracted from S. hermonthica seedlings at various times during haustorial development (45 min, 1.5 h, 3 h, 5 h and 7.5 h), as well as from water treated control seedlings. 2-D gel analysis improved the resolution of the proteins since it separates polypeptides both by charge and by mass (figure 5.2).
F ig u r e 5.2
Tw o-dim ensional PAGE separations of water soluble proteins from untreated and 2,6-DM BQ treated seedlings. (A) protein pattern before treatment, (B) untreated for 7.5 h, (C) treated for 45 min, (D) 1.5 h, (E) 3 h and (F) 5 h. Numbered lines indicate 2,6-DMBQ induced proteins.
Open circles represent proteins that became less abundant during treatm ent. M olecular w eight markers (kD) are along the left side.
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Comparison of the 2-D PAGE profiles of water soluble proteins from treated and untreated seedlings revealed that while most proteins
rem ained unaffected, some appeared de novo or increased in abundance and others disappeared or decreased in abundance in response to
2,6-DM BQ. Protein samples from the control treatm ents, that is
germ inated seedlings at time zero and seedlings incubated in w ater for 7.5 h, produced the same 2D pattern indicating that there were no time dependent changes over the course of the experiment (Figure 5.2 A and B). However, treatment of seedlings with 2,6-DMBQ for only 45 min
(Figure 5.2 C) caused the appearance of one new protein (13). After 1.5 h in 2,6-DMBQ (Figure 5.2 D) two proteins became relatively more
abundant (9 and 10) and remained at these elevated levels throughout the rest of the experiment. Also, a sharp decrease in the abundance of one protein (12) was observed at this time. Longer exposures to 2,6-DMBQ caused a further gradual decrease in this polypeptide. At 3 h (Figure 5.2 E) there was a slight increase in the abundance of two polypeptides (2 and 3) and a transient increase in the abundance of the novel
polypeptide (13) which had returned to its original level relative to the other proteins by 5 h (Figure 5.2 F). At 5 h treatment, the polypeptides 2 and 3 had become more evident along with five others (4,5,6,7 and 11), in addition the novel appearance of two more proteins ( 1 and 8) was
protein com plem ent between 5 h and 7.5 h (result not shown) treatm ent of Striga seedlings with 2,6-DMBQ.
In this study it has been demonstrated that changes in protein synthesis occurred in S. hermonthica seedlings within 45 minutes of treatm ent with 2,6-DMBQ (the appearance of protein 13). This is before any m orphological changes can be detected (refer to figures 3.5 and 3.6). A transient increase in this new polypeptide (13) was seen around 3 h which corresponds to the first noticeable signs of radicle swelling of treated seedlings. More proteins appeared and increased in abundance by 5 h, at which time radicles are noticeably hairy.
These proteins may have been involved in haustorial
developm ent. A number of proteins, including protein 12, were of high relativ e abundance after the germ ination treatm ent. H ow ever, only protein 1 2 was noticeably reduced in abundance over the short time
course of the experiment in response to 2,6-DMBQ treatment. Thus, protein 1 2 may play a special role in metabolism during haustorial
d e v e lo p m e n t.
Some o f the proteins that accum ulated during haustorial induction may have done so as a direct consequence of exposure of seedlings to 2,6-DMBQ. However it is expected that most of the changes in protein abundance were a consequence of haustorial in itiation
were sufficient to initiate haustorial development. Results in Chapter 4 (section 4.2.1) dem onstrated that seedlings which were rem oved from 2,6-DM BQ at 40 min and placed in water, later showed radial expansion and haustorial hair developm ent although radicle elongation continued leaving a "ring" of hairs midway along the radicle of the seedling. This dem onstrated the short exposure time to 2,6-DMBQ which was required in order to initiate cell differentiation. Thus, the changes in protein synthesis observed at 45 min may be involved in the actual initiation of h a u s to ria l d ev elo p m en t.
As described in Chapter 1, Chang and Lynn (1986) have suggested that 5 rr/g a-d e riv e d enzyme activity around the radicle is responsible for releasing 2,6-DMBQ from the surface of host sorghum roots. Thus, in vivo, the entire S tr ig a seedling would not be exposed to an haustorial inducer. The protein extractions described above were carried out on the entire seedling including the contents of the seed coat (Cotyledons and
aleurone; W orsham and Egley, 1990). Therefore, it should be considered that exposure of the entire seedling to 2,6-DMBQ may have induced protein-synthetic changes in tissues not normally exposed to an h a u sto ria l in d u cer.
Similar work with S. asiatica (Wolfe and Timko, 1992) also dem onstrated the appearance of new proteins during haustorial induction. However, the 2-D gel patterns of S. asiatica and S.
W olf and Timko (1992) extracted total labelled protein from only 50 seedlings whereas water soluble proteins from 50 |ig (approximately 9 000 seedlings) were extracted in the experim ents presented here.
5 .2 .2 RN A e x tr a c tio n s .
Using the large scale haustorial method described in C hapter 3, 500 mg batches of S. hermonthica seeds were used to provide germinated and haustorially induced seedlings. Total RNA was extracted from the seedlings following the method modified from D raper et al. (1987) described in Chapter 2.
The total RNA from the Striga seedlings was always found to be light brown in colour. This could be attributed to the presence of triisopropyl naphthalene sulphonate (TNS) in the extraction buffer (H. Logan, pers. comm.). The total RNA invariably contained a sticky com ponent that would not resuspend in water. This was thought to be due to polysaccharide contam ination which is a common problem in plant RNA extractions (Draper et al. 1987).
It was possible to measure the yield and purity of the S t r i g a total RNA by measuring the optical density (O.D.) at 260 and 280 nm (see section 2.6.5). Pure RNA has an (OD) ratio of 2:1 at 260:280 nm. The yield was estimated by assuming that a 40 |ig ml-i RNA has an O.D. of one at 260 nm. Total RNA extracted from S tri ga seedlings had O.D. ratios
extractions with low protein contam ination had been achieved despite the viscosity and brown discolouration of the total RNA preparations. The yields which were obtained (594-758 |ig g-i seed weight) com pared well with other RNA preparations from host root m aterial (section 7.2.5) where yields of 350 |Xg g-i f. wt. were obtained from sorghum root.
However, RNA yields were calculated on the Striga seed weight prior to preconditioning and the fresh weight after im bibition and germ ination may be considerably higher, thus the RNA yields may be overestim ates on a fresh weight basis.
Agarose gel analysis was routinely carried out to determ ine the integrity of the total RNA. The gels were submerged in running buffer containing ethidium bromide at 1 pg ml-i. Ethidium brom ide
intercalates with the RNA molecules. Thus, the RNA can be visualised under ultraviolet light because ethidium bromide fluoresces. On
separation, two strong staining bands were observed which corresponded to the ribosom al RNA that makes up the 25S and IBS ribosom al subunits. The 25S ribosomal RNA band stained more strongly than the IBS RNA band when visualised under ultraviolet light. Where degradation has occurred this staining pattern is reversed since 25S ribosomal RNA is characteristically degraded to an IBS-like species. The total RNA from Stri ga seedlings was always found to be undegraded as dem onstrated by the agarose gel presented in figure 5.3.
T a b le 5.1
Yield and purity of total RNA extractions on Striga seedlings. Results are the mean of 4 extractions, +/- SD.
S am p le |ig total RNA g-i d. wt. seed purity OD260/280 G e rm in a te d In d u c e d 594 +/- 242 758 +/- 296 1.84 +/- 0.16 1.87 +/- 0.1 2 5 S - 2 5 S - I 8 S _ F ig u re 5.3
(A) 1 jig total RNA from S. hermonthica was separated on a 1% agarose gel, stained with ethidium bromide and visualised under UV light. Brightly fluorescing ribosomal bands are present indicating the integrity of the RNA.
(B) 0.5 |ig mRNA from control (1) and haustorially induced (2) S. her mon thica seedlings was separated as for the total RNA. The absence of ribosomal bands demonstrates the purity of the mRNA. 25S and 18S in
The total RNA extractions were consistently higher from the
haustorially induced seedlings. The ribosomes consist of ribosom al RNA, together with many proteins. They coordinate the interplay of transfer RNA, mRNA and protein in the complex process of protein synthesis. Thus, the increased levels of total RNA in haustorially induced S t r i g a seedlings may indicate that increased levels of protein synthesis were occurring in these seedlings compared to germinated seedlings.
Various methods were used in an attempt to purify the
polyadenylated mRNA from the total RNA. Colbert (1983) suggested the use of an oligo (dT) cellulose column (as described in section 2.6.6a) to purify mRNA. However, in this study such an approach was unsuccessful, perhaps due to the viscosity of the total RNA sample. The use of the param agnetic bead purification procedure (described in section 2.6.6b) was very successful. The best ratio of total RNAibead volume was found to be 250 }Xg:600|il providing mRNA recoveries of 0.5-2% (estim ated from the O.D. at 260 nm). Increasing the total RNA quantity relative to the bead volume did not improve the recovery of mRNA. The yield was good, considering that mRNA accounts for only 1-2% of total RNA. Agarose gels were used to assess the purity of the mRNA. Separation of the mRNA dem onstrated the absence of any ribosomal bands (see fig. 5.3). This indicated that the mRNA was not contaminated with ribosom al RNA. The size range of the mRNA recovered from Striga seedlings was estim ated
using a standard RNA marker separated on the same gel as the mRNA. The mRNA was found to range in size from approximately 8 kb to 0.5 kb. This indicated that full-length transcript mRNA had been recovered.
This was the first report of successful RNA extractions from S t r i g a seedlings. The success of the RNA extractions was dependent on the quantity of tissue generated in the large-scale haustorial induction system which had been developed earlier (see section 3.2.3).
5 .3 .3 m R N A T r a n s la tio n s
Cell-free translations as described in section 2.7.7 were carried out in order to analyse the variety and differences between the pools of mRNA, isolated as described above (5.2.2) from germ inated and h au sto rially induced S. hermonthica s e e d lin g s .
5 pi 35S-labelled cell-free translation products were separated in 10% acrylamide SDS slab gels. For a higher resolution of the translation products, a further 10 pi of samples were separated by 2-D
(lEF/SDS-PAGE) gel electrophoresis. Visualisation of the translation products was by direct autoradiography for SDS-PAGE gels, and by fluorography for the 2D gels (figure 5.4).
kD 9 7 - 6 9 - 4 6 - 3 0 - 1 4 - kD 2 0 0- 9 7 - 6 9 - 4 6 - 3 0 — 1 4 - / ° \ 7
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O ’ * F i g u r e 5. 4Cell-free translations using substrate mRNA from germinated (A) and haustorially induced (B) S. hermonthica seedlings. After the germination treatment, seedlings were treated with water or IQ-^M 2,6-DMBQ for 7.5 h at 33°C at 180 rpm. Seedlings were collected and ground to a fine powder in liquid nitrogen for total RNA extraction. Purified mRNA was used in cell-free translations conducted at 25oC for 1 h. Translation products were separated by SDS-PAGE (above) and 2-D gel electrophoresis (below).
SDS-PAGE analysis of the in vitro translation products, presented in figure 5.4, showed that proteins ranging in size from 14-100 kD were synthesized from the two pools of mRNA, dem onstrating the biological activity of the purified mRNA from the Striga tissue. However, it was not possible to discern any differences between the two protein patterns because of the number of synthesised products. Thus 2-D electrophoretic separation of the cell-free translation products was carried out to enable a more detailed analysis of the translation products.
2-D electrophoresis was more successful in separating the translation products. Analysis of the 2-D gel patterns of translation products (Fig. 5.4) revealed that several changes had occurred after incubating the seedlings in 2,6-DMBQ for 7.5 h. Most of the proteins synthesised in vitro rem ained unaltered after haustorial induction. However, some spots showed a decrease in abundance (1,8 and 9), and new ones appeared (2,3,4,5,6 and 7). The changes in protein abundance that occurred represent the decrease or novel appearance of mRNA
species resulting from haustorial induction. H austorial developm ent was complete at the time of RNA extraction, so the different levels of mRNA observed were probably a result of haustorial developm ent, and may direct the synthesis of proteins involved in radial cell expansion or h au sto rial hair developm ent.
explained by increased gene transcription. However, it m ust be
rem em bered that increased stabilisation or translatability o f the mRNA may cause the same effect. For example, an increase in the rate of both transcription and stabilisation of the mRNA accounts for the
accum ulation of ovalbumin by steroid hormones in chick oviduct (M cKnight and Palmiter, 1979). Further research is required to
investigate the reasons for the increase/decrease of particular translation p r o d u c ts .
The differential accumulation of translatable mRNA in haustorially induced seedlings may account for some or all of the changes in the w ater soluble protein profile during haustorial induction (section 5.2.1). However, direct comparisons of the 2D gel patterns of water soluble proteins and translation products could not be made because the mRNA used in the cell-free translations codes for the total protein of the S t r i g a se ed lin g s.
The success of the in vitro translations showed that the mRNA was biologically active and could be used for cDNA synthesis. M essenger RNA (4 jig) from germinated and haustorially induced S. hermonthica
seedlings respectively was used as the template for two cDNA synthesis reactions (A. Fanigliulo, pers. comm.). The cDNA library constructed using RNA from haustorially induced seedlings could be used to isolate clones representing mRNAs that are induced during haustorial induction by differential screening. Differential screening is commonly used to
isolate tissue specific and developmentally regulated cDNA sequences (H erdenberger et a i , 1990, Herskey and Quail 1984).
A scheme for differential screening of the library containing haustorially specific cDNA was developed (figure 5.5). This involved plating out the library and making duplicate plaque filter lifts. A plaque is the clear area in a continuous sheet of E. coli cells caused by lam bda phage-induced lysis of E. coli and the DNA within the plaque can be directly transferred to nylon filters. One of the filters would then be probed with 32p_iabelled mRNA (or cDNA) from germinated seedlings, and one with 32p_iabelled mRNA (or cDNA) from haustorially induced
seedlings. Those plaques which gave a positive signal with both probes would represent cDNAs derived from mRNA species that are abundant in germ inated and haustorially induced seedlings. However, those plaques which gave a positive signal with the 'haustorial' probe, but not with the 'germ inated' probe should correspond to plaques containing haustorial specific cDNA. Haustorial specific plaques could then be isolated from the m aster plate. The haustorial specific cDNA could be recovered for further analysis by sequencing. The sequence could be com pared to known sequences which may pinpoint the function o f these gene
products. This would help to show whether the induced genes caused or resu lted from haustorial developm ent.
F ig u r e 5.5
cDNA library m ade from RNA o f haustorially induced Striga seed lin gs is plated out
Duplicate plaque lifts are taken
Hybridise with labelled cDNA from germ inated Striga
mRNA
nitrocellulose m em brane
Hybridise with labelled cDNA from haustorially induced
Striga mRNA
Wash and autoradiograph
Autoradiograph on X-ray film
Example o f a plaque containing a se q u en ce abundant in both germ inated and haustorially induced seedling mRNA
Example o f a plaque containing a se q u en ce th at is abundant in haustorially induced mRNA but
M olecular research on A. tumefacienslhost interaction has
provided a model for the transduction of host derived phenolic signals to 'turn on' the bacterium's vir genes (Winans et al.y 1984, Clarke, Leigh and Douglas, 1992). Should this work continue, it may be possible to identify the mode of action of 2,6-DMBQ in S. hermonthica.
5.4 SUMMARY
This study set out to investigate the biochem istry and m olecular