INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

Microtubules are found in all eukaryotic cells and are involved in maintenance of cell shape, mitosis, and a variety of other morphogenic events (6). At the core of these multimeric structures lie heterodimeric tubulin filaments made up of two very similar - and -tubulin proteins. Microtubules are never static, and their constant assembly and disassembly involving tubulin filaments and various accessary proteins is an integral part of their function. Despite the highly conserved nature of - and -tubulins, sufficient differences exist between cell types and taxa to provide opportunities for selective interference with the interaction between these tubulin proteins. Consequently, several natural products, as well as some synthetic chemicals, have been developed as antitubulin agents and are used in cancer therapy and as herbicides, antihelminthics, or fungicides (5).

Benzimidazoles are a group of broad-spectrum systemic fungicides which interact with tubulin (1), and especially -tubulin. Resistance is a major practical problem in the use of these fungicides; it is caused by point mutations in -tubulin which replace Glu₁₉₈ with either Ala, Val, or Gly, or which replace Phe₂₀₀ with Tyr (4). The same mutations are also encountered in veterinary medicine where parasitic nematodes have become resistant to treatment with benzimidazoles (9). At least eight other mutational sites within -tubulin confer resistance in laboratory strains, but these mutations have not so far been encountered in field strains. What is particularly interesting about benzimidazole resistance is its link with increased sensitivity to phenylcarbamates, which do not affect wild-type strains (7). This negative cross-resistance offers a durable way to combat resistance, and a benzimidazole-phenylcarbamate mixture has been used commercially as an antiresistance strategy against grey mold (Botrytis cinerea) in several crops, with varying levels of success. Double-resistant mutants can occur, but their frequency is often low so that performance of the mixture is not affected. Understanding the molecular changes in -tubulin should help the mixture to be used more effectively and, since negative cross-resistance often occurs where there are target site changes, to provide generic solutions for using similar mixtures as antiresistance strategies.

Normal levels of tubulin proteins in filamentous fungi are low, and purification has not been possible. Overexpression of - and -tubulin genes in Aspergillus nidulans allowed purification of assembly-competent tubulin, but the drug paclitaxel (Taxol) was required to promote their assembly into microtubules (12). Cloning of fungal tubulin genes coupled with heterologous expression offers a way to produce large amounts of native - and -tubulins in Escherichia coli, which is free of microtubule accessory proteins since these are not found in bacteria. Unfortunately, expression of otherwise soluble tubulin proteins in E. coli has not provided information about their conformation, largely because they aggregate and accumulate as insoluble inclusion bodies. In vitro expression in rabbit reticulocyte lysates may overcome this problem, as well as providing chaperones required for correct tubulin folding (2), but protein levels are low (10 nM) and insufficient for further analysis. The three-dimensional structure of mammalian tubulin was produced recently through electron crystallography, and, with the resolution at 3.2 Å, individual amino acids interacting with the bound anticancer drug paclitaxel could be assigned correctly (10). Structure-activity studies involving benzimidazole chemistry and fungal strains carrying different point mutations which confer benzimidazole resistance have also provided some insight into the binding site (5), but individual amino acids involved have not been identified in this way with certainty.

In an attempt to overcome these difficulties, we have explored the expression of a fungal -tubulin from Rhynchosporium secalis (barley leaf blotch) in E. coli. High-level expression of soluble -tubulin can be achieved as a fusion with a maltose binding protein (MBP), and the binding of benzimidazole (carbendazim) and phenylcarbamate (diethofencarb) fungicides to both wild-type and mutant proteins correlates well with biological activity. This suggests that at least the fungicide binding site folds correctly.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

This work was carried out under license PHL 19/2176 (11/1996), issued by the United Kingdom Ministry of Agriculture, Fisheries and Food, to construct genetically modified plant pests.

Preparation of -tubulin cDNA. Total RNA was extracted from a 7-day-old culture of R. secalis 1130 (8) with guanidine thiocyanate and an RNA isolation kit (Stratagene, La Jolla, Calif.). This RNA (3 µg) was used to construct an adapter-ligated cDNA library according to the protocol in the Marathon kit (Clontech, Palo Alto, Calif.). The presence of a full-length -tubulin cDNA gene was confirmed by sequencing 5' and 3' RACE (rapid amplification of cDNA ends) products, and the whole gene was amplified from this cDNA library with primers (35-mers) designed for the 5' and 3' ends of the coding sequence with EcoRI (5') and HindIII (3') restriction sites. The EcoRI restriction sequence was positioned so that the cDNA clone could be inserted into expression vectors in frame with the fusion protein sequence. The Expand High Fidelity PCR system (Boehringer, Mannheim, Germany) was used to amplify fragments, and their sequences were confirmed with Sequenase (Amersham International, Little Chalfont, United Kingdom) after cloning into pUC18.

Expression in E. coli. -Tubulin was overexpressed in several vectors (see Table 1) in E. coli XL1-Blue. Expression as a fusion with the MBP (pMAL-c2) was induced with IPTG (isopropyl--D-thiogalactopyranoside) as described in the manufacturer's protocol (New England Biolabs, Beverly, Mass.) and as a Histidine-6 fusion in pTrcHis-B (Express system) according to the manufacturer's instructions (Invitrogen, San Diego, Calif.). The gene was also inserted into pRSET, and expression was induced with IPTG followed by infection with M13 T7 phage (5 PFU per bacterial cell). The sequence was also inserted into the pKK-223-3 expression vector (Pharmacia Biotech, Uppsala, Sweden). In all cases, cells were harvested from 50-ml broth cultures when the optical density at 600 nm reached 1.0. To analyze total protein, cells from a 1.0-ml culture were collected by centrifugation and lysed in sodium dodecyl sulfate (SDS), and the protein was separated by SDS-8 to 10% polyacrylamide gel electrophoresis. Quantification was achieved by densitometry after staining with Coomassie blue stain (see Table 1). To determine the solubility of recombinant protein, remaining cells were broken by sonication and debris was removed by centrifugation (14,000 × g for 20 min). Expression was again analyzed by SDS-8 to 10% polyacrylamide gel electrophoresis, and the presence of -tubulin in soluble fractions was confirmed by Western blotting using a mouse monoclonal antibody against sea urchin (Stronglyocentrotus purpuratus) -tubulin (T-5293; Sigma Chemical Co., St Louis, Mo.). High levels of expression were within those claimed by the manufacturer and reflect the strong induction by IPTG of the Ptac promoter. Whereas over 90% of the MBP--tubulin fusion protein remained in the soluble fraction after centrifugation, the opposite was the case with the pRSET fusion and at least 90% sedimented with cell debris.

Binding of radiolabelled fungicides to fusion proteins. The soluble MBP--tubulin fusion protein extract was adjusted to 15% glycerol and either used immediately or, if stored at 4°C, filtered through a 1.25-µm-pore-size filter (Millipore, Watford, United Kingdom). Crude lysates (1.0 ml; 4 to 5 mg of total protein) were incubated for 60 min at 4°C in the presence of 0.005 mM MgCl₂, 0.1 M KCl, and 0.25 µCi of either [¹⁴C]carbendazim (140 µCi/mmol) (Amersham International) or [¹⁴C]diethofencarb (700 µCi/mmol) (kindly supplied by M. Fujimura, Sumitomo Chemical Company, Osaka, Japan). Bound and unbound fungicides were separated on a Sephacryl HR-300 column (45 by 2 cm) using 0.05 M sodium phosphate buffer (pH 6.8), 0.005 M MgCl₂, and 0.1 M KCl and a flow rate of 1.0 ml min¹. Fractions (3.0 ml) were collected, and their protein contents were determined by the microplate version of the Bradford procedure (Pierce Chemicals, Peoria, Ill.). Radioactivity was counted in a liquid scintillation spectrometer.

Nucleotide sequence accession number. The genomic sequence determined in this study has been deposited in the EMBL no. 1 database under accession no. X81046.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Only vector pKK-223-3 failed to generate useful expression of -tubulin; the other vector systems all yielded considerable amounts of the protein (Table 1). However, only the fusion with the MBP produced sufficient soluble protein to allow analysis of its fungicide binding properties. The MBP itself (40 kDa) bound no fungicide, although some radioactivity eluted in all extracts along with a low-molecular-weight peptide just ahead of the unbound fungicide. Carbendazim bound to the wild-type (Glu₁₉₈) fusion protein but not to the corresponding resistant (Gly₁₉₈) protein (Fig. 1). The reverse was the case for diethofencarb, which bound to the Gly₁₉₈ fusion protein and not to the wild-type protein. Thus, binding correlates well with negative cross-resistance, which is a feature of the biological activity of these two fungicide groups, and indicates that the -tubulin folds correctly, even as a fusion protein. The approximate stoichiometry of binding was calculated with knowledge of the specific activity of each fungicide and by determining the protein in fractions corresponding to the MBP fusion protein. In three separate experiments involving the wild-type (Glu₁₉₈) fusion protein, the ratio of protein to fungicide bound ranged from 1:1 to 1.5:1. For the resistant protein (Gly₁₉₈), because of the higher specific activity of diethofencarb, much less fungicide bound to the fusion protein.

View this table: [in this window] [in a new window]   TABLE 1.   Expression of -tubulin fusion proteins in E. colia

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Effects of wild-type protein (Glu198) and resistant mutant protein (Gly198) on binding of fungicides to MBP--tubulin fusion proteins. To obtain disintegrations per minute values, 1.0 ml of each 3.0-ml fraction was counted.

Although the fungicide binding sites fold correctly, we have not determined whether the entire -tubulin also folds in the right way. Benzimidazole-resistant mutants generated in the laboratory indicate that other regions of -tubulin than amino acid codons 198 to 200 are involved in fungicide binding, and if these regions were not folding correctly this would have influenced carbendazim binding to the wild-type protein. -Tubulin is a GTPase, and several domains interact with the nucleotide. Binding of GTP would also indicate whether the protein was folding correctly, but as yet we have not attempted experiments which would first require removal of the endogenous GTP from the extract prior to binding studies.

These results complement earlier genetic (3) and sequencing (4) studies and confirm that benzimidazole and phenylcarbamate fungicides do indeed bind to -tubulin. Replacement of a polar amino acid at codon 198 with a small neutral one clearly alters the protein sufficiently to influence binding, although exactly which part of -tubulin interacts directly with the fungicides remains to be established. We are currently exploring the effects of other point mutations on fungicide binding, as well as the influence of -tubulin, in order to establish the extent to which these fungicides affect the interface between the two monomers and formation of the heterodimer. Although linking of -tubulin to a soluble protein reduced the extent of aggregation, it was not possible to cleave the MBP fusion with factor Xa. Further purification and concentration of the fusion protein on an amylose affinity column according to the manufacturer's protocol resulted in aggregation. Under conditions described in the Express manual, the Histidine-6 fusion bound poorly to the nickel resin and elution with just 50 mM imidazole failed to achieve any purification. The strongest expression was achieved with pRSET, although all of this protein was pelleted following centrifugation and was assumed to be in inclusion bodies. Careful renaturing of the inclusion bodies may well be the best approach to securing a better three-dimensional structure of fungal -tubulin.