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L. 212.121.; 91.1....(1. { In ’41., .l.;.. r .. ,.... ‘46P” lllllllllllllllllll 1 This is to certify that the dissertation entitled Characterization of beta-tubulin gene from benomyl— resistant field strains of Venturia inaequalis presented by Harrie Koenraadt has been accepted towards fulfillment of the requirements for Doctoral degreein Botany & Plant Pathology 4. fl... Maj-cry... Date September 22, 1992 MSU is an Affirmative Action/Equal Opportunity Insrirution 0-12771 a» LIBRARY Michigan State University 0, PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution CHARACTERIZATION OF THE BETA-TUBULIN GENE FROM BENOMYL- SENSITIVE AND BENOMYL-RESISTANT FIELD STRAINS OF VENTURIA INAEQUALIS BY Harrie Koenraadt A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1992 ABSTRACT CHARACTERIZATION OF THE BETA-TUBULIN GENE FROM BENOMYL- SENSITIVE AND BENOMYL-RESISTANT FIELD STRAINS OF VENTURIA INAEQUALIS BY Harrie Koenraadt This study was conducted to determine the molecular basis for widely differing levels of resistance to the benzimidazole fungicide benomyl and sensitivity to the N- phenylcarbamate fungicide diethofencarb in Venturia inaegualis, the causal organism of apple scab. The beta- tubulin gene from a field strain with very high resistance (VHR) to benomyl and from a sensitive field strain were cloned and characterized by sequence analysis. The polymerase chain reaction (PCR), subcloning of the amplified DNA, and subsequent sequence analysis were used to characterize a part of the beta-tubulin gene from field strains with low resistance (LR), medium resistance (MR), or high resistance (HR) to benomyl. Point mutations that altered codon 198 for glutamic acid to lysine and alanine codons were associated with HR and VHR phenotypes, respectively, while alteration of codon 200 for phenylalanine to tyrosine was associated with the MR phenotype. Allele-specific oligonucleotide analysis was used to evaluate point mutations in 20 additional field strains of V. inaequalis and the expected mutations were found in all resistant strains but one from Chile. Sequence analysis revealed that an alternate codon for tyrosine was present in the strain from Chile. Characterization of the beta-tubulin gene from benomyl-resistant field strains of other plant pathogenic fungi revealed that the codon changes were identical to those in V. inaequalis. A comparison of the codon conversions in the beta-tubulin gene of field strains of plant pathogenic fungi with laboratory-induced benomyl-resistant mutants in Aspergillus nidulans and Neurospora crassa showed that mutations conferring field resistance represent a subset of the mutations recovered in laboratory experiments. Transformation experiments were initiated to investigate whether mutations in codons 198 and 200 were directly responsible for resistance to benomyl and sensitivity to diethofencarb. Site-directed mutagenesis was performed on pBT6, a plasmid that contains the beta-tubulin gene of N. crassa and confers resistance to benomyl due to a mutation in codon 167. Codon 167 was altered to the original wild- type codon, and codons 198 or 200 were altered to obtain the mutations observed in V. inaegualis. Transformants of N. crassa were recovered that exhibited resistance to benomyl and increased sensitivity to diethofencarb. The transformation experiments indicate that amino acids 198 and 200 of beta-tubulin play a key role in resistance to benzimidazoles and sensitivity to structurally related N- phenylcarbamates. The deduced amino acid sequence of beta-tubulin of V. inaequalis showed extensive similarity with beta-tubulin genes of species of different phyla and classes. Microheterogeneity was observed at several positions throughout the sequence but not in position 198. Phylum- specific microheterogeneity was observed around position 200, a position in which a substituted amino acid conferred resistance to benomyl. Therefore, phylum-specific microheterogeneity in the beta-tubulin might be the basis of the selective toxicity of benzimidazole and N- phenylcarbamate compounds. DEDICATION To my father Piet Koenraadt, who unfortunately just passed away before finishing my degree, and my mother An Koenraadt- Meijer. ii ACKNOWLEDGMENTS I want to thank professor Alan Jones for his guidance and support during my education at MSU. Working in his group, in which both molecular biology and field work are areas of interest, was quite enjoyable and stimulating. The trips to Traverse City and the visits to the Blue BirdTM will be remembered. I am also grateful to my committee members, Dr. John Wilson, Dr. Ray Hammerschmidt, and Dr. Shauna Somerville. Especially, the opportunity to work in Shauna's lab helped me lot during the start of this project. Her constructive discussions and reviewing of manuscripts were very helpful to me. I want to thank Dr. Gus de Zoeten for his interest in my work and some personal advice during my stay at MSU.» I also express my appreciation for Hanneke Hoekstra, Karl Pearson, Gail Ehret, Chien-Shun Chiou, Lizhe Liang, Patty McManus, and many other friends who made my stay more enjoyable. Finally, I want to thank my family and some good old friends in the Netherlands for their emotional support. iii TABLE OF CONTENTS LIST OF TABLES.. .................................... .. LIST OF FIGURES................................. ...... GENERAL INTRODUCTION AND LITERATURE REVIEW ............ LITERATURE CITED.... ......... .. ..... . ................. PART I CHARACTERIZATION OF MUTATIONS IN THE BETA-TUBULIN GENE OF BENOMYL-RESISTANT FIELD STRAINS OF VENTURIA INAEQUALIS AND OTHER PLANT PATHOGENIC FUNGI ABSTRACT .............................................. INTRODUCTION...... .................................... MATERIALS AND METHODS ........ .......... ............... Fungal strains .......... . ..... ........... ........ Bacteria and plasmids............................ Cloning the beta- tubulin gene from Venturia inaequalis........ ........................... Sequence analysis of the beta- tubulin gene from Venturia inaequalis............... .............. Rapid cloning of the beta- tubulin gene with the polymerase chain reaction ..... ..... .............. RESULTS ............. . ......................... . ....... Cloning and sequence analysis of the beta-tubulin gene from Venturia inaegualis .................... Characterization of beta-tubulin DNA fragments from other fungal species........................ DISCUSSION. ........................................... LITERATURE CITED ...................................... iv Page vii viii 15 21 23 25 25 26 26 28 28 32 32 38 41 48 PART II THE USE OF ALLELE-SPECIFIC OLIGONUCLEOTIDE PROBES TO CHARACTERIZE RESISTANCE TO BENOMYL IN FIELD STRAINS OF VENTURIA INAEQUALIS ABSTRACT.....0.0.00.0000000000000000000000000000000000 INTRODUCTION......... ........ ... ..... O. ....... . ....... MATERIALS ANDMETHODS...00.0.0.0...O....0.0.0.0.....0. Fungal Strains.....0.0.......................0.0. DNA isolation and polymerase chain reaction...... Allele-specific oligonucleotide probes........... Allele-specific oligonucleotide analysis......... Direct characterization of alleles in apple scab leSionS ..... .O... ..... . ...... ... ..... ..... ....... RESULTS... ................ ..... ....................... Allele-specific oligonucleotide analysis......... Direct characterization of alleles in apple scab lesions.... ....... ......... ......... . ..... .. ..... DISCUSSION. ........................................... LITERATURE CITED ...................................... PART III MUTATIONS IN CODON 198 OR 200 OF THE BETA-TUBULIN GENE CONFER RESISTANCE TO BENOMYL AND CODON 198 ALSO CONFERS SENSITIVITY TO DIETHOFENCARB ABSTRACT... ............................... ............ INTRODUCTION ............ ..... ...... . ........... ....... MATERIALS AND METHODS...................... ...... ..... Fungal and bacterial strains and plasmids.. ...... Construction of plasmid pTyr ........ ...... ....... Site-directed mutagenesis in the beta-tubulin gene............................................. Transformation of Neurospora crassa.............. Molecular characterization of transformants by Southern analysis................................ Growth response of transformants to fungicides... 52 54 56 56 56 57 57 59 61 61 66 68 72 75 76 78 78 78 79 81 83 84 RESULTS..........00...... ..... ......O............... Site-directed mutagenesis in the beta-tubulin gene........ ..... .............................. Transformation of Neurospora crassa............ Growth of transformants on medium amended with fungicides..................................... DISCUSSION..... ....................................... LITERATURE CITED. ....... ........ ........ . ..... ... ..... APPENDICES......... .......... . ..... .... ............... APPENDIX A: SELECTIVITY OF BENZIMIDAZOLES AND N- PHENYLCARBAMATES IS BASED ON MICROHETEROGENEITY OF BETA-TUBULINS REVIEW.................................... ........... . LITERATURE CITED ......... . ...... ...................... APPENDIX B: RECOMMENDATIONS ........................... Rapid detection of point mutations............. Is microheterogeneity the basis for selectivity of benzimidazoles and phenylcarbamates? .......... Transformation of Aspergillus nidulans. .......... LITERATURE CITED ............ . ......................... vi 85 85 85 86 88 92 93 95 108 113 113 114 115 117 LIST OF TABLES Table PART I Point mutations and deduced amino acid substitutions in the beta-tubulin gene for field strains of several plant pathogenic fungi with resistance to benomyl and sensitivity to diethofencarb (NPC) and methyl-N-(3,5-dichlorophenyl)carbamate (MDPC) ..... Deduced amino acid substitutions in the beta-tubulins of laboratory—induced mutants with resistance to benzimidazole fungicides. ..... . PART II Benomyl resistance phenotypes of field strains of Venturia inaequalis from different geographic regions and hybridization of a series of allele- specific oligonucleotide (ASO) probes to beta— tubulin DNA from each strain ...................... PART III Plasmid designations and codon alternations in the beta-tubulin gene of Neurospora crassa...... ...... APPENDIX Comparison of the deduced amino acid residues in beta-tubulin gene of Venturia inaequalis and several organism at positions that might be critical for resistance to benzimidazoles and related compounds ........... .. .................... Page 36 40 62 82 105 LIST OF FIGURES PART 1 Figure Page 1. Nucleotide sequence of the beta-tubulin gene from the benomyl-sensitive strain WC of Venturia inaequalis. The nucleotide sequence is numbered from the ATG initiation codon (numbers appear above the nucleotides). The deduced amino acid sequence is indicated by the three letter amino acid code below the nucleotides (numbers are given below the amino acid sequence). The six intervening sequences are indicated by IVS 1—6. Putative 5' and 3' splice junction and lariat formation sequences in IVS 1-6 are shown in bold. A potential polyadenylation sequence in the 3'-f1anking region of the gene is underlined. Annealing sites for primers A-D (see text) are underlined. A BamHI site, directly downstream of primer A, BfrI, EcoRI, and BamHI sites in primers B, C, and D, respectively, were used for the directional cloning of amplified DNA, and are shown above the nucleotide sequence. Three non- homologous nucleotides in primer D, as shown in the BamHI sequence, were used to generate a new BamHI site in amplified DNA ....................... 33 PART II A, Sequence of allele-specific oligonucleotide (ASO) probes for Venturia inaequalis. The A80 probes for medium (MR), high (HR), and very high resistance (VHR) to benomyl differ from the beta— tubulin DNA for sensitive (S) and low resistance (LR) strains by one nucleotide (arrows). B, Sequence of codons 195 to 203 of the beta- tubulin gene from benomyl-sensitive field strains of 10 plant pathogenic fungfi. Under conditions of high stringency, the ASOS' probe will be removed from its complementary anti—sense strand sequence from fungi other than V. inaequalis due to one to three mismatches (underlined) in the third position of codons ................................ 58 viii Allele-specific oligonucleotide analysis with amplifigd Beta-tubulin DNA of Venturia inaequalis. The A80 7 probe hybridized to DNA on dot blots in panel A and B, and the blots were subsequently washed at a temperature (WT) of low stringeEcy (A) or ogfiimum stringency (B). The ASOMR , ASOH and ASOV probes were hybridized to DNA on the blots in panels C, D, and B, respectively. The blots were subsequently washed at optimum stringency. Amplified beta-tubulin DNA, from sensitive, low, medium, high, and very high resistant strains was applied to each blot in column S, LR, MR, HR, and VHR, respectively. S strains (from top to bottom): B7, MSU-18, and WC; LR strains: MINNS 118, MITCHELL 94-1, and MSU-20; MR strains: MAINE 4, MAINE 8, and SIS—16; HR strains: RI, I—23, and RH-4; and VHR strains: TU86R2, 75-26, and KV3C.... 64 PART III Growth response, after 1 day at 30 C, of parent strain arg-3 (30300) A and benomyl-resistant transformants of Neurospora crassa on Vogel's medium (upper left quadrant of each plate) and Vogel's medium amended with benomyl (upper right quadrant), diethofencarb (lower left quadrant, or benomyl and diethofencarb (lower left quadrant). The petri dish in the upper left position contains the parent strain, while the middle and left petri dishes contain transformants with pPHEMR DNA. The petri dishes in the lower row contain transformants with pPHE DNA .................... 87 APPENDIX A Chemical structures of carbendazim, diethofencarb (NPC), and methyl-N—(3,S-dichlorophenyl)carbamate (MDPC)(A). Comparison of three dimensional images of carbendazim with diethofencarb (B) and MDPC (C) .......................................... 98 Comparison of the deduced amino acid sequences of the beta-tubulin gene of Venturia inaequalis with those of beta—tubulin genes from selected species of several taxonomically distant phyla. The amino acid sequence of V. inaequalis is shown at top. Amino acids differing from the beta-tubulin of V. inaequalis are shown for each beta—tubulin of other species, while identical amino acids are indicated by dashes. The deduced amino acid sequence of the highly variable C—terminal peptide is shown for each beta-tubulin gene ............... 101 ix GENERAL INTRODUCTION AND LITERATURE REVIEW Modern agriculture relies heavily on the use of pesticides. Herbicides and insecticides are widely used to control weeds and arthropods. Bactericides and fungicides are applied on a regular basis to control bacterial and fungal diseases of crops, respectively. A large variety of drugs are used to safequard animal, including human, health. Pesticide and drug resistance, defined as the development of a stable and heritable adjustment by a cell to external or endogenous toxic or inhibitory agents (54), poses a serious threat to agricultural production and animal health. This review will mainly deal with resistance to benzimidazole compounds and tubulin, the target site of benzimidazole compounds. A few additional examples of resistance to pesticides and drugs are discussed to emphasize that resistance to pesticides and drugs exists among a wide variety of harmful organisms and has major implications for our society. Protectant-type fungicides were introduced more than a century ago to control fungal diseases. The effectiveness of protectant fungicides did not change despite extensive usage over several decades. The multisite mode of action of most of these protectant fungicides prevented fungi from developing resistance. An explanation for the lack of resistance development is that a mutation in one target site will not alter the sensitivity of the mutant because several sites must be altered for resistance to develop (26). The introduction of systemic fungicides with a site-specific mode of action had several advantages over fungicides with a multisite mode of action. Disease control was obtained with fewer applications of relatively low rates of site-specific fungicides. However, the rapid development of resistance among plant pathogenic fungi to fungicides with a site- specific mode of action such as benzimidazoles, carboxamides, acylalanines, and sterol biosynthesis inhibitors have seriously compromised disease control (61). Resistance to pesticides and drugs is not restricted to fungi. Resistance to antibiotics is a common phenomenon among clinical and plant pathogenic bacteria (8, 10, 48). A resurgence of tuberculosis, incited by Mycobacterium tuberculosis, is due to the rapid spread of antibiotic- resistant strains. Multiple-resistance in M. tuberculosis, prevents the efficient control of tuberculosis. For example, there are strains that are resistant to 11 different antibiotics (13). Resistance is also common among parasites of animals, including human parasites. For example, the control of malaria, caused by Plasmodium falciparum, is compromised by the rapid spread of strains with resistance to antimalaria compounds such as chloroquine and quinine (45). An additional problem is that eradication of the parasite by vector control has become impossible because of insecticide resistance in mosquitoes (45). Failure of chemotherapy among people with cancer is caused by the rapid development of resistance in tumor cells to several cytotoxic drugs including colchicine, vincristine, vinblastine, and taxol (3, 4). Characterization of resistant mutants of bacteria, fungi, insects, plants, and vertebrates revealed that some biochemical resistance mechanisms are of great importance and evolved in a wide variety of organisms. Mechanisms such as increased rate of detoxification, decreased rates of uptake, and changes in target sites are known to confer resistance to pesticides and drugs (26, 48). Several excellent reviews have described the importance of fungicide (15, 26, 61), bactericide (10, 48), insecticide (38, 40, 49), herbicide (27) and drug resistance (40, 48). Detoxification of pesticides and drugs is a common resistance mechanism among insects and bacteria. Overproduction of enzymes such as hydrolyses, mixed function oxidases, glutathione transferases, and dichlorodiphenyltrichloroethane (DDT)-ases are involved in the increased detoxification of insecticides (49). Overproduction of detoxifying esterases in aphids, Drosophila, and mosquitoes are associated with DNA amplification of esterase—encoding genes (17). Detoxification of antibiotics by specific enzymes such as acetyltransferases and phosphotransferases is often used by antibiotic producers to avoid suicide (14). Clinical and plant pathogenic bacteria use the same strategy and produce enzymes with similar functions (8, 13, 48). Decreased rates of uptake of pesticides and drugs is a resistance mechanism that occurs in a wide variety of organisms (1, 48, 67). Prevention of access of the antibiotics beta-lactams, aminoglycosides, and tetracyclines to the target site through alternation of permeability or efflux is common among several clinically important bacteria (48). Overproduction of a high-molecular weight plasma membrane glycoprotein in tumor cells is associated with multidrug resistance (mdr) to functionally unrelated cytotoxic agents that are used in clinical chemotherapy, such as the benzimidazole compound nocadazole, colchicine, taxol, and actinomycin D (3). The P-glycoproteins appear to function as energy-dependent transport proteins and are encoded by a family of genes that are evolutionary conserved among a wide variety of organisms. Resistance of P. falciparum to antimalarial agents such as quinine and chloroquine is also associated with amplification and overexpression of P—glycoprotein-encoding genes (1). Mdr genes are also involved in resistance to insecticides (67) and might be involved resistance to sterol biosynthesis inhibitors of fungi (29). Multi-drug resistance has been widely reviewed in recent years (1, 3, 4, 20, 28). A change in target-site is an alternate mechanism for reducing the sensitivity of organisms and cells to pesticides and drugs. Resistance to insecticides such as dieldrin, DDT, and organophosphates are caused by mutations that alter target sites (22, 49). Failure of 3'-azido-3'- deoxythymidine (AZT) therapy in patients with acquired immunodeficiency syndrome (AIDS) is associated with point mutations in the reverse transcriptase gene of the HIV-1 virus (62). Some bacterial strains became resistant to antibiotics by modification of target sites, a strategy that is also exploited by antibiotic producing organisms (14, 48). Resistance to fungicides such as benzimidazoles, phenylamides, and carboxamides is also attributed to modifications of the target sites in fungi (61). The mode of action of benzimidazole compounds and the development of resistance to these compounds will be discussed in more detail. Benzimidazole compounds, introduced in the 1960's and early 1970's, are widely used as fungicides and anthelmintics. Thiabendazole (TBZ), a safe broad-spectrum anthelmintic, was released in 1961 to control parasitic nematodes in animals, including humans. Reports of resistance followed several years after the introduction of TBZ (40). Resistance to benomyl, a benzimidazole compound with high toxicity to Ascomycetous fungi emerged two to three years after the release of benomyl for crop protection. Control of apple scab, caused by Venturia inaequalis, failed after three years of extensive and exclusive application of benomyl (32). Resistant strains were grouped into four classes (low, LR; medium, MR; high, HR; and very high resistant, VHR) based on the growth response of the strains on media amended with benomyl. Genetic analysis revealed that mutations in a single gene were responsible for widely differing levels of resistance to benomyl among strains of V. inaequalis (36, 55, 60) and V. pirina (57). Compounds of N-phenylcarbamate and N- phenylformamidoxime chemistry were found that inhibited benomyl-resistant but not benomyl—sensitive strains of several plant pathogenic fungi (37, 31). Negatively correlated cross-resistance, the phenomenon that resistance to one compound is associated with increased sensitivity to a second compound, was observed among strains with very high resistance (VHR) but not strains with low resistance (LR), medium resistance (MR), or high resistance (HR) to benomyl. VHR strains exhibited sensitivity to the N-phenylcarbamate fungicide diethofencarb, while S, LR, MR, and HR strains were insensitive. The N-phenylcarbamate fungicide MDPC was only active against strains with the HR and VHR phenotype (33, 56). Because some benomyl-resistant strains are not controlled by N-phenylcarbamate fungicides it is unlikely that these compounds will provide a solution to the problem of widely spread resistance to benomyl. Studies on the mode of action of benzimidazole compounds were extensively reviewed by Davidse (15) and Lacey (40). Initial studies showed that nuclear division of sensitive strains of fungi was disrupted by benzimidazole compounds. Cytological observations revealed that the effects of benzimidazoles on arresting nuclear division in fungi were similar to the cytological effects of colchicine. Colchicine, a secondary plant metabolite with very high toxicity to animals, interferes with microtubule assembly. In fact, tubulin was once classified as the colchicine binding protein (66). Binding experiments with radiolabelled carbendazim, the toxic conversion product of benomyl, and crude mycelial extracts of benomyl-sensitive strains of A. nidulans suggested that sensitivity was correlated with binding of carbendazim to tubulin. Decreased binding of carbendazim to tubulin from a laboratory-induced benomyl resistant strain of A. nidulans supported the hypothesis that microtubule assembly was inhibited by benomyl (15, 16). Decreased binding of mebendazole to tubulin was also reported for benzimidazole- resistant isolates of Haemonchus contortus and Trichostrongylus colubriformis (42, 51). Characterization of the beta-tubulin gene of a strain of Neurospora crassa with resistance to benomyl revealed a point mutation that altered codon 167 from phenylalanine in a sensitive strain to tyrosine in a resistant strain. Transformation experiments provided evidence that this point mutation was responsible for resistance to benomyl (50). Point mutations in codons 6 and 165 were found in beta- tubulin genes from benomyl-resistant strains of A. nidulans that were previously used in the carbendazim binding experiments by Davidse (16, 34). Mutations in other codon positions of the beta-tubulin gene were found in several additional benomyl resistant strains of organisms such as A. nidulans and Saccharomyces cerevisiae (35, 64). A mutation in codon 198 of the beta-tubulin from N. crassa conferred resistance to the benzimidazole compound benomyl and sensitivity to the N-phenylcarbamate compound diethofencarb. It was hypothesized that diethofencarb could bind to the slightly altered carbendazim binding site (24). Characterization of benzimidazole resistant mutants of the nematode Caenorhabditis elegans also revealed point mutations in the ben-l beta-tubulin gene (18). In conclusion, compelling evidence from studies with a variety of organisms indicates that point mutations in certain critical codons of the beta-tubulin gene are a major cause of resistance to benomyl. A change in a single critical amino acid in the beta-tubulin appears to prevent the interference of carbendazim with tubulin assembly in such mutants. It can not be ruled out that alternative mechanisms might be involved in resistance to benomyl. The opportunistic pathogenic fungus Candida albicans is naturally insensitive to benomyl. However, benomyl-sensitive transformants were obtained when a beta-tubulin gene of C. albicans was expressed in S. cerevisiae by heterologous gene-replacement (58). The results of the study suggest that an alternative mechanism such as degradation or active exclusion of benomyl might be involved in the natural resistance of C. albicans (58). Characterization of the beta-tubulin gene from a benomyl resistant strain of Epichloe typhina revealed no alteration in the beta-tubulin coding sequence and only a rearrangement of the 5' flanking region of the beta-tubulin gene (7). In organisms with several beta-tubulin genes, the gene that encodes for a beta-tubulin that is sensitive to a benzimidazole compound may be deleted. Some benzimidazole- resistant mutants of C. elegans were shown to have lost the ben-l gene without affecting their viability under laboratory conditions (18). It is unknown how carbendazim and several other related drugs interfere with tubulin assembly. Carbendazim, colchicine, and most other drugs induce depolymerization of microtubules, while taxol induces stabilization of microtubules (30, 47). Microtubules are hollow, tubular structures, comprised of a series of 13 protofilaments. Protofilaments are the products of the polymerization of tubulin, a dimer of alpha- and beta-tubulin. Microtubules are an essential part of the cytoskeleton of eukaryotic cells and play a vital role in numerous functions in cells such as maintenance of cell shape, intracellular transport, chromosome movement, cell motility, cellular secretion, and nutrient absorption (15, 40). The tubulin dimer is the major component of microtubules, but several additional proteins are associated with the organelle. A few of these microtubules-associated proteins (MAPS) have been 10 characterized (11), but the function of most MAPs is unknown. Several models have been postulated to explain the dynamics of microtubules (21). The current model of dynamic instability (46) states that the end of a microtubule is never in equilibrium. Most microtubules grow at a slow, steady rate, but a small fraction of the microtubules shorten, or depolymerize, at a fast rate at the same time (21). The energy source for polymerization of tubulin comes from the hydrolysis of guanosine triphosphate (GTP). Microtubules with a GTP cap are thought to be stable and favor polymerization, while these with a GDP (guanosine diphosphate) core would be unstable and favor depolymerization of microtubules. The differential stability of microtubules with a GTP cap and a GDP core may be related to different conformations of the microtubule ends (21). The interference of antimitotic agents on tubulin-GTP interaction is reviewed by Correia (12). Binding of colchicine to tubulin induces a change in the secondary structure of tubulin and weak GTPase activity (12). The fact that benzimidazole compounds and colchicine are competitive inhibitors (40) suggests that benzimidazoles compounds also might induce GTPase activity, but this hypothesis has not been supported by experimental evidence. A large number of alpha- and beta-tubulin genes have been cloned and characterized from a wide variety of organisms. In addition, a number of gamma-tubulin genes have recently 11 be characterized from diverse organisms such as A. nidulans, Drosophila, and humans (5). Cloning of tubulin genes was facilitated by the existence of significant homology between tubulin genes of different species. A comparison of the deduced amino acid sequences of tubulin genes revealed that microheterogeneity increases when organisms are evolutionarily more distant (9, 23, 41, 63). Homology is not only restricted within subclasses of tubulin but also exists between subclasses. Sequence comparisons of the deduced amino acid residues of 160 tubulin genes by Burns, in which the highly divergent C-terminal was excluded, revealed 63% (alpha/beta), 51% (alpha/gamma), and 59% (beta/gamma) identity (5). Alpha- and beta-tubulins are usually encoded by multigene families. Lower eukaryotic organisms such as fungi have only one or two beta-tubulin genes, but plants and vertebrates have several additional tubulin genes (23, 43, 50). For example, at least six alpha-tubulin genes and nine betaftubulin genes are expressed in Arabidopsis thaliana (39, 59). Post-translational mechanisms including acetylation, reversible tyrosination, glutamylation, and phosphorylation are thought to create additional heterogeneity among tubulins (23, 63). The significance of the existence of alpha- and beta-tubulin isotypes within an organism is a subject of speculation. The multitubulin hypothesis states that the tubulin isotypes are functionally different and can be used for different functions in a 12 particular microtubule array (25). Evidence for the multitubulin hypothesis is not abundant (41, 63). The nematode C. elegans contains three beta- tubulin genes, ben-l, tub-1, and mec-7. Expression of the mec-7 gene for beta-tubulin is required for the formation of unusual 15-protofilament microtubules in C. elegans and mec- 7 mutants are touch insensitive (53). However, mutants with a deleted ben-l gene are viable and coordinated (18). In fungi, it appears that tubulin isotypes can complement each other. A gene disruption study revealed that the two unusual divergent beta-tubulin genes of A. nidulans are interchangeable and expression of only the benA or benC beta-tubulin did not interfere with the viability of mutants (44). An alternative for the multitubulin hypothesis is that the existence of multiple tubulin genes allows a more optimal expression of certain tubulin genes over time and in different tissues. The tubC gene of A. nidulans is expressed only during conidiation (65). Tissue-specific and developmentally regulated expression of beta-tubulin isotypes has been reported for numerous higher eukaryotic organisms (23, 63). A thorough understanding of the interactions of tubulin isotypes with MAPS might eventually explain why there are different tubulin isotypes. The exact mechanism of selective activity of benzimidazole compounds among different organisms is unknown. In binding studies, helminth tubulin had a higher affinity for 13 anthelmintics benzimidazoles than mammalian tubulin (51). Tubulin of different species also exhibited different affinities for colchicine (6). Separation of mammalian tubulin isotypes by immunoaffinity chromatography, and subsequent binding studies with colchicine revealed differential affinity of tubulin isotypes to colchicine (2). The differential affinity for benzimidazoles and colchicine among tubulins within and between different species is not surprising since even single amino acid substitutions in the beta-tubulin gene from A. nidulans interfere with the binding of carbendazim (16, 34). Therefore, it is likely that microheterogeneity among beta-tubulin of different organisms might be involved in selective toxicity of benzimidazoles and colchicine. Elucidation of the three-dimensional structure of tubulin will be necessary to understand how benzimidazoles, N- phenylcarbamates, and several other drugs interfere with tubulin assembly. In addition, selective drugs could be designed to exploit microheterogeneity among tubulins from different organisms (19, 68). However, X-ray analysis of tubulin is not possible because of a failure to grow suitable crystals and NMR (nuclear magnetic resonance) analysis is precluded by the high molecular weight and the tendency to self-aggreate. Constraints on the possible tertiary structure of tubulin subunits have been extensively reviewed by Burns (5) who compared a large number of tubulin genes. Surface features of tubulin were predicted by 14 examination of insertions, deletions, and the variability of amino acid residues that exist among alpha- and beta- tubulins. Epitope mapping data and putative GTP-binding motifs were compiled and evaluated to obtain complementary information about surface features of tubulin. The question of whether mutations are present in the beta- tubulin of benomyl-resistant strains of V. inaequalis and other plant pathogenic fungi is addressed in Part Iiof this thesis. A study to elucidate the molecular basis of the differential resistance to benomyl and sensitivity to diethofencarb is also described in Part I. Part II describes a method to rapidly characterize point mutations in the beta-tubulin gene of V. inaequalis. Part III describes a study to show that mutations in codons 198 and 200 are directly responsible for differential resistance to benomyl and increased sensitivity to diethofencarb. Finally, a hypothetical model for the basis of selectivity of benzimidazole and N-phenylcarbamate compounds is discussed in the appendix of this thesis. " aim; s-u-o- LITERATURE CITED 1. Balzi, E., and Goffeau, A. 1991. 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Physiol. 40:149- 161. may...“ .- .12.". ..-- -- PART I CHARACTERIZATION OF MUTATIONS IN THE BETA-TUBULIN GENE OF BENOMYL-RESISTANT FIELD STRAINS OF VENTURIA INAEQUALIS AND OTHER PLANT PATHOGENIC FUNGI ’..lw- i—n ~' 6.... ABSTRACT All benomyl-resistant field strains of Venturia inaequalis, V. pirina, Monilinia fructicola, Sclerotinia homoeocarpa, and six species of Penicillium, except those with low resistance to benomyl, were found to contain a single base-pair mutation in their beta-tubulin gene that resulted in an amino acid substitution in beta-tubulin. In V. inaequalis, codon 198, which encodes glutamic acid in a sensitive strain, was converted to a codon for alanine in a strain with very high resistance, to a codon for lysine in a strain with high resistance, or to a codon for glycine in a strain with medium resistance to benomyl. Codon 200 for phenylalanine was converted to a codon for tyrosine in a second strain of V. inaequalis with medium resistance to benomyl. Among field strains of other fungi, 14 had a glutamic acid to lysine, alanine, or valine substitution at position 198 and three had a phenylalanine to tyrosine substitution at position 200. Among seven benomyl-resistant strains with sensitivity to the N-phenylcarbamate fungicide diethofencarb, all had a glutamic acid to alanine or glycine substitution at position 198. A comparison of the codon changes in the beta-tubulin gene of field strains with laboratory-induced benomyl-resistant mutants of model fungi 21 22 showed that mutations conferring field resistance represent a small subset of the mutations recovered in laboratory experiments. INTRODUCTION Benomyl-resistant Venturia inaequalis (Cke.) G. Wint. isolates were detected shortly after intensive and exclusive use of benomyl in Michigan apple orchards (12) and in most countries where benzimidazole fungicides were used to control apple scab. In V. inaequalis, resistance to benomyl was attributed to mutations in a single gene with different alleles conferring widely different levels of resistance (29, 34). Sensitivity of V. inaequalis to N- phenylcarbamates, a group of mitosis-inhibiting fungicides, was found to be negatively correlated with very high resistance (VHR) to benomyl. Sensitivity to the N- phenylcarbamate fungicides diethofencarb and methyl-N-(3,5- dichlorophenyl) carbamate (MDPC) was also attributed to a single gene and it was postulated that the VHR allele for resistance to benomyl was identical with the allele for sensitivity to diethofencarb (11, 30). The characterization of laboratory-induced resistant mutants in model organisms like Drosophila, Aspergillus, and Arabidopsis has become a common approach for studying resistance mechanisms to pesticides (5, 8, 13, 14, 15). In binding studies (3), beta-tubulin from a benomyl-resistant strain of A. nidulans exhibited reduced affinity for 23 24 carbendazim, the toxic conversion product of benomyl. Later, molecular characterization of the beta-tubulin gene of benomyl-resistant mutants of A. nidulans (13, 14), Neurospora crassa (6, 26), and Saccharomyces cerevisiae (35) provided evidence that point mutations in this gene were responsible for resistance to benomyl. A point mutation in the beta-tubulin gene was also found in a benomyl-resistant strain of N. crassa with increased sensitivity to diethofencarb and it was postulated that diethofencarb could bind to the altered carbendazim binding site (6). Whether these same mutations were responsible for the development of benomyl-resistant plant pathogenic fungi has not been determined. This study was conducted to determine the molecular basis for resistance to benomyl in field strains of V. inaequalis with widely different levels of resistance to benomyl and sensitivity to N-phenylcarbamates. The basis for benomyl resistance in field strains of other plant pathogens was investigated to establish whether mutations associated with resistance in these fungi were identical to the mutations found in V. inaequalis. Mutations in the beta-tubulin gene of field strains were compared with mutations reported for laboratory-induced strains of model fungi to determine whether the same mutations were involved in field and laboratory-induced resistance to benomyl. MATERIALS AND METHODS Fungal strains. The single-spore isolates of V. inaequalis were from a worldwide collection of field strains previously characterized in studies on the inheritance of resistance to benomyl and N-phenylcarbamates (11, 34). Isolates of several species of Penicillium were provided by D.A. Rosenberger, New York State Agricultural Experiment Station, Cornell University, Geneva, NY; those of V. pirina Aderhold by E. Shabi, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; those of Monilinia fructicola (G. Wint.) Honey by A.L. Jones; and those of Sclerotinia homoeocarpa F.T. Bennett by J.M. Vargas, Michigan State University, East Lansing, MI. All of the strains were field isolates. The cultures were maintained on potato dextrose agar (PDA)(Difco Laboratories, Detroit, MI). The reaction of the strains to benomyl (Benlate 50 WP; DuPont de Nemours & Co., Wilmington, DE) and to MDPC and diethofencarb (Sumitomo Chemical Company, Ltd., Takatsukasa Takarazuka, Japan) was determined by transferring pieces of mycelium to PDA amended with benomyl at 0, 0.5, 5, and 50 mg/L, MDPC at 2.5 mg/L, or diethofencarb at 0.5 mg/L. The strains were then classified as established in previous 25 26 studies on benomyl resistance (11, 16, 29, 30, 31) into one of the following phenotypes: sensitive (S), no growth on 0.5 mg/L benomyl; low resistant (LR), growth on 0.5 mg/L, but not 5 on mg/L; medium resistant (MR), growth on 5 mg/L, but not on 50 mg/L; high resistant (HR), slow growth on 50 mg/L; very high resistant (VHR), rapid growth on 50 mg/L. Strain I-26 of V. inaequalis, previously reported as atypical for MR isolates due to its unusual sensitivity to diethofencarb (10, 28), was assigned the phenotype MR“. Bacteria and plasmids. Escherichia coli strain LE 392 (24) was used as a host for phage replication. Plasmids pUC19 and pUCBM20 (Boehringer Mannheim, Indianapolis, IN) were used for cloning experiments and were propagated in E. coli strains DH5alpha and HB101 in the presence of ampicillin at 50 mg/L. Cloning the beta-tubulin gene from Venturia inaequalis. Agar plugs with actively growing mycelium of the benomyl- sensitive strain WC or the very high resistant strain KV3C were fragmented in sterile distilled water with a tissue grinder (Cat. No. 7727; Corning Glass Works, Corning, NY) and added to 200 ml of potato dextrose broth (PDB)(Difco Laboratories, Detroit, MI) in 1 L flasks coated with dichlorodimethylsilane. The cultures were incubatedon a rotary shaker at 100 rpm for 3 wk at about 20 C. The mycelium was harvested by filtration of medium through Miracloth (Calbiochem Corp., La Jolla, CA). The mycelium (5-10 g wet weight) was rinsed with distilled water, dried 27 between paper towels, and then frozen with liquid nitrogen in a mortar. Frozen mycelium was ground to a powder, suspended by vortexing in 50 ml of lysis buffer (100 mM LiCl, 50 mM NazEDTA, 1.0% SDS, 10 mM Tris-HCl, pH 7.4) with 20 ug/ml proteinase K (Boehringer Mannheim, Indianapolis, IN) and then incubated at 50 C for 1 h. The DNA was purified by phenol extraction and ethanol precipitation. The pellet was resuspended in 4 ml TE buffer (10 mM Tris- HCl, pH 7.4, 1 mM EDTA), and mixed with 4.2 g CsCl and 0.17 ml ethidium bromide (10 mg/ml). The solution was centrifuged in a Sorvall TV-865 rotor (Du Pont Company, Wilmington, DE) for 14 h at 220,000 x g. The DNA was partially digested with Sau3A (Boehringer Mannheim, Indianapolis, IN) and size fractionated on a 10 to 40% sucrose gradient. DNA fragments 16-20 kb in size were ligated into dephosphorylated, BamHI-digested, EMBL3 arms according to the instructions of the manufacturer (Promega, Madison, WI). The ligation mixtures were packaged in Vitro with Packagene lambda DNA packaging system (Promega, Madison, WI) and the resulting phage were used to transfect E. 0011 LE 392. The beta-tubulin gene cloned from Erysiphe graminis f.sp. hordei (32) was used as a probe to screen the libraries. Two internal HindIII DNA fragments, encoding codons 22 to 428 of the E. g. hordei beta-tubulin gene, were labeled with alpha-32P-dCTP (Du Pont, Boston, MA) using the random primed labeling method (4). Hybridization was performed in a 28 solution of high ionic strength (6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ug/mL denatured salmon sperm DNA)(21) at 55 C for 12 h. Filters were then washed twice in each of the following solutions: 2X SSC at 20 C for 5 min, 2X SSC, 1% SDS at 55 C for 30 min, and 0.1x SSC at 20 C for 30 min. Recombinant phage DNA that hybridized with the heterologous beta-tubulin probe was digested with the restriction enzymes BamHI, BglI, EcoRI, SalI, XbaI, and XhoI (Boehringer Mannheim, Indianapolis, IN) and subjected to Southern analysis (33). Sequence analysis of the beta-tubulin gene from Venturia inaequalis. Unidirectional deletions of the beta-tubulin gene of strain WC and strain KV3C were obtained using the Erase-a-Base system (Promega, Madison, WI) according to the protocol of the manufacturer. Sequencing of insert DNA was performed by the dideoxy chain termination method (28) with Sequenase (United States Biochemical, Cleveland, OH). Alkaline-denatured double-stranded plasmid DNA was used as template in the sequencing reactions (36). Both strands of the coding region and 300-bp of the upstream and downstream flanking regions of the beta-tubulin genes were sequenced. Rapid cloning of the beta-tubulin gene with the polymerase chain reaction. Actively growing mycelium was fragmented with a tissue grinder and added to 50 ml PDB in siliconized 250 ml flasks. The cultures were shaken at 100 rpm at about 20 C for 1-5 days. DNA samples were prepared as described above except that the final step, CsCl equilibrium density 29 centrifugation, was omitted. The polymerase chain reaction (PCR) and then sequence analysis of the cloned DNA fragment were used to rapidly characterize the sequence of beta- tubulin DNA from a variety of strains. Genomic DNA was prepared from each strain of V. inaequalis and subjected to PCR, in which a 22-mer oligonucleotide A (5'- CAAACCATCTCTGGCGAACACG) and a 22-mer oligonucleotide B (5'- TGGAGGACATCTTAAGACCACG) were used as primers. Primer A was identical in sequence to codons 22 to 28 and primer B was complementary in sequence to codons 359 to 365 of the beta- tubulin gene of V. inaequalis (Fig. 1). With these primers a 1,188-bp fragment of the beta-tubulin gene was amplified. A BamHI restriction site, immediately downstream of the annealing-site of primer A, and a BfrI restriction site in primer B (Fig. 1), were used to subclone the amplified beta- tubulin DNA. Genomic DNA prepared from each of the other fungal species was subjected to PCR by using two generic beta-tubulin primers. Constraints on primer design were that the amplified DNA had to contain codons 167 and 241, codons in which mutations were associated with resistance to benomyl (26, 35), and that the primers had to anneal to a conserved region with minimal variation in the sequence. A 24-mer oligonucleotide C (5'-GAGGAATTCCCAGACCGTATGATG) and a 28-mer oligonucleotide D (5'-GCTGGATCCTATTCTTTGGGTCGAACAT) were chosen as generic beta-tubulin primers. Primer C was nearly identical in sequence to codons 157 to 164 and primer D was 30 complementary in sequence to codons 293 to 300 of the beta- tubulin gene (Fig. 1). PCR with these primers was expected to amplify a 436-bp fragment of the beta-tubulin genes. Primers C and D contained an EcoRI and BamHI restriction site, respectively, to facilitate subcloning of amplified DNA (Fig. 1). The primers were synthesized in the Macromolecular Structure Facility, Department of Biochemistry, Michigan State University, East Lansing. Reactions were performed in a thermal cycler (Perkin- Elmer, Norwalk, CT) using the Repliprime DNA amplification system (Du Pont, Boston, MA) according to the manufacturer's procedures. Negative controls were run in all the amplification reactions to detect contamination. In reactions involving primers A and B, 35 cycles were performed for each reaction as follows: 94 C, 1 min; 55 C, 1 min; and 72 C, 2 min. In reactions involving primers C and D, the annealing temperature was reduced from 55 to 50 C. Amplification products were analyzed for the expected 1,188- bp or 436-bp fragments by agarose gel electrophoresis in 1X TBE buffer (0.1 M Tris-HCl, 0.1 M boric acid, 0.02 mM EDTA, pH 8.3). Following electrophoresis, the DNA was visualized with ethidium bromide. The amplified sequences were precipitated, digested with the appropriate restriction enzymes, directionally subcloned into pUC19 or pUCBM20, and the resulting plasmids were used to transform E. coli strain DH5 or HBlOl. A 300-bp DNA fragment was sequenced with custom designed 31 beta-tubulin-specific sequencing primers to characterize codons 164 to 264 in the subcloned DNA fragments from LR, MR, and HR strains of V. inaequalis. The subcloned, 436-bp DNA fragments from the other fungal species were sequenced entirely. DNASIS (Hitachi America Ltd., San Bruno, CA) software was used to analyze and compare the sequences. Open reading frames (ORF) found in the sequences were compared with previously characterized beta-tubulin genes of other fungi to determine whether the sequences were beta-tubulin sequences. Comparisons of the deduced amino acid sequences from sensitive and benomyl-resistant strains were used to determine the amino acids that were associated with resistance to benomyl. The codon number assignments of partially characterized beta-tubulin sequences of fungi other than V. inaequalis were based on homology with other beta-tubulin genes (1). The codon substitutions observed in the field strains were compared with changes reported for laboratory-induced mutants with resistance to benomyl (6, 13, 14, 26, 35). RESULTS Cloning and sequence analysis of the beta-tubulin gene from Venturia inaequalis. The genomic libraries of the S and VHR strains containing 1 x 104 and 2 x 104 clones, respectively, were screened with the radiolabelled beta- tubulin clone from E. g. hordei. DNA from two and eight clones of the S and VHR libraries, respectively, hybridized strongly with the heterologous beta-tubulin DNA probe. In Southern analysis (data not shown), single bands of BamHI, BglI, XbaI, and XhoI-digested DNA fragments from the lambda clones were found to hybridize with the E. g. hordei beta- tubulin probe. Additional restriction analysis, in combination with Southern analysis, suggested that a 7.0 kb XhoI DNA fragment of the clones contained the entire beta- tubulin, and this DNA fragment was subcloned from one clone of each library into pUC19. Nucleotide sequence analysis of DNA cloned from the S and VHR strains of V. inaequalis revealed the presence of seven exons, encoding a protein of 446 amino acids, and six intervening sequences (IVS 1-6) (Fig. 1). Comparison of the deduced amino acid sequence from the cloned sequence of V. inaequalis and the beta-tubulin genes from E. g. hordei (32), N. crassa (26), and A. nidulans (benA) (22) showed 98, 32 33 Fig. 1. Nucleotide sequence of the beta-tubulin gene from the benomyl-sensitive strain WC of Venturia inaequalis. The nucleotide sequence is numbered from the ATG initiation codon (numbers appear above the nucleotides). The deduced amino acid sequence is indicated by the three letter amino acid code below the nucleotides (numbers are given below the amino acid sequence). The six intervening sequences are indicated by IVS 1-6. Putative 5' and 3' splice junction and lariat formation sequences in IVS 1-6 are shown in bold. A potential polyadenylation sequence in the 3'-flanking region of the gene is underlined. Annealing sites for primers A-D (see text) are underlined. A BamHI site, directly downstream of primer A, BfrI, EcoRI, and BamHI sites in primers B, C, and D, respectively, were used for the directional cloning of amplified DNA, and are shown above the nucleotide sequence. Three non-homologous nucleotides in primer D, as shown in the BamHI sequence, were used to generate a new BamHI site in amplified DNA. 34 U7 TGluGluA M) m m 4 mm m 2 mm 6 mm mm mm mm 6mm mam mw mm . . awn mam mmm 58mm mm mam mm mm mew 0mm MW gm mm mam m an _ mm mm mm mm mm may mm mm M69 6% mm mm A mm mm mm my 8 mm nmm «a 88% mm mm mm 6 mm M mm mm m mm mm mm mm 1a» mama mm mmm m m firm we mu saw 8 mam mm mm ms Ce: my 1mm my a m a was new mm mm 8 am“ mm mm mm m mm mm mum _ . . w a m am an an mam mm mm mam mm A mm 84 my 6 mm mm mm mm mm mm mm m mam Ann an A m mmm was “88 may my mm mm awn M mm m% 8mm st mm mm m an man 86 ms was mm mm m m nu mum m mm mm mm mam 5mm w W“ mm m4 1. m% A A“ M "W MM mm m m_ at m. mm mm 14 m mm a am a a _ mm 888 mm my mmm my mu a. mm mm mm m N am. ma my mm 98% mm mm mm Tm mm mm m6 mma _ um mm m mum my 8mm mm m mm as m m W @b R Eh W MW WW mm 1mm mm WWW W HEW W mm 4 mums mm mam my mm m mm m mm mm Emu m a m m _ mg was mm mm 8% ma mu 863 mm mm a e um mum mm mm 8mm mm new L mm we mu m mm mm mm mm mm mm mam n6 mww mm mm 1 mm m em» 8 mm mm man mm mm“ my wma mm m @883 z: m mm mm 8mm mm mm 8 mm ms mm mm m m _ m um mm mm can men mum an my mm mm my mm m m 6_ 6 mm mm was. um mm as was was mm mm mm a m a. mm mm mmmm mm mu mm mm mm my 8 m m m .6 .T _ any mm as“ mime as 8 mm mm mm mm G W” mine 8 mam “mm any we mm mwm mu me mm mm 188 mm m6 mm mm mm mm. mm mm “Wm mm mm m 2 we mmm me mm mm mm 1mmm as am m 6 mm mm a m away a _ mum mm mm m6 mm 8. ma mmm m6 mm mmm a a a m m was an m6 mmm ma new mu mm mm m4 mm 1mm . 4 _ m mm mm mm mm as mm mma mm mm mm mm mm mm 6 mm mm «a mm mm «mm mm ms was new sue mm mm mm. .nm_ mwm mm awn ms nu mm mm me mm .86 mum m mm mm_ vs mm mm mm mum any mm M. an 1 mm m mm .mm mm mm mm mm mm mm cam mm wuss 888 mm a 1mm1 man mm mm mm mmm mm mm as mm” mwmam mm 41mm mm mm mm WAWMTMWAMWMUAWMWAUUWMUWAUMWWWMMW 95 HD 88 35 35 mm mm 85 NB 35 96, and 96% homology, respectively. In addition, the positions of IVS 1-6 in the cloned sequences of V. inaequalis were identical with the positions of IVS 1-6 in the beta-tubulin gene from E. g. hordei and N. crassa (data not shown). The nucleotide sequence for the beta-tubulin gene of V. inaequalis was submitted to the Genbank data base under accession number M 97951. In Southern analysis with 8911, XbaI, and XhoI digested genomic DNA of strain KV3C, only single bands of DNA with a size of 20, 30, and 7.0-kb respectively, hybridized with the cloned beta-tubulin DNA from V. inaequalis. Only a single codon change was detected when the structural beta-tubulin gene from the VHR strain KV3C was compared with the nucleotide sequence of the beta-tubulin gene cloned from the 8 strain WC. Codon 198 for glutamic acid (GAG) in the S strain was replaced by a codon for alanine (GCG) in the VHR strain (Table 1). The 300-bp DNA fragments of PCR amplified DNA from LR, MR, MR”, and HR strains of V. inaequalis showed complete or nearly complete sequence identity with the corresponding region in the sequenced beta-tubulin gene from 8 strain WC. The MR, MR’, and HR strains of V. inaequalis exhibited single base-pair mutations, but no mutations were observed in the LR strain. The MR strain contained a single base- pair mutation converting codon 200 from phenylalanine (TTC) in the benomyl-sensitive strain to a codon for tyrosine (TAC). The MR' and HR strain contained mutations converting 36 TABLE 1. Point mutations and deduced amino acid substitutions in the beta-tubulin gene for field strains of several plant pathogenic fungi with resistance to benomyl and sensitivity to diethofencarb (NPC) and methyl-N-(B,S-dichlorophenyl)carbamate (MDPC) Phenotype Amino acids Fungal species Codon in position and strains Benomyl MDPC NPC substitution 198 199 200 Venturia inaequalis wc sa ISb IS none Glu Thr Phe MINNS ll8 LR IS IS none Glu Thr Phe I-26 MR- 5 s GAG to GGG Gly Thr Phe MAINE 8 MR IS IS TTC to TAC Glu Thr Tyr RH-4 HR 3 IS GAG to AAG Lys Thr Phe KVBC VHR S S GAG to GCG Ala Thr Phe Venturia pirina IL-7 S IS IS none Glu Thr Phe IL-8 S IS IS none Glu Thr Phe IL-2 MR IS IS TTC to TAC Glu Thr Tyr IL-S VHR S S GAG TO GCG Ala Thr Phe IL-6 VHR S S GAG TO GCG Ala Thr Phe Monilinia fructicola SHANE s -C IS none Glu Thr Phe CB-2 HR - IS GAA to AAA Lys Thr Phe Penicillium puberulum CO-4 S IS IS none Glu Thr Phe CO-67 HR 8 IS GAG to AAG Lys Thr Phe 00-164 VHR S S GAG to GCG Ala Thr Phe Table l (Cont'd). 37 Penicillium digitatum DI-V4 S IS IS none Glu Thr Phe DI-M18 HR S IS GAG to AAG Lys Thr Phe DI-M13 HR 8 IS GAG to AAG Lys Thr Phe Penicillium expansum FIX-99 S S IS none Glu Thr Phe EX-132 LR IS IS none Glu Thr Phe EX-301 HR S IS GAG to GTG Val Thr Phe EX-24 VHR S S GAG to GCG Ala Thr Phe Penicillium italicum IT-7E S S IS none Glu Thr Phe IT-7G MR IS IS TTC to TAC Glu Thr Tyr IT-7M HR 8 IS GAG to AAG Lys Thr Phe Penicillium aurantiogriseum 80-198 LR IS IS none Glu Thr Phe 80-147 MR IS IS TTC to TAC Glu Thr Tyr 80-126 HR S IS GAG to AAG Lys Thr Phe 80-298 VHR S S GAG to GCG Ala Thr Phe Penicillium viridicatum VI-227 8 IS IS none Glu Thr Phe VI-77 HR S IS GAG to AAG Lys Thr Phe Sclerotinia homoeocarpa BEN-S S - IS none Glu Thr Phe BEN-R HR - IS GAG to AAG Lys Thr Phe aBenomyl, MDPC, and NPC phenotypes were determined by growing the strains on PDA amended with benomyl at O, 0.5, 5, and 50 mg/L, MDPC at 2.5 mg/L, or NPC at 0.5 mg/L, respectively. Sensitive (S), no growth with benomyl at 0.5 mg/L ; low resistant (LR), growth on 0.5 mg/L, but not 5 on mg/L; medium resistant (MR), growth on 5 mg/L, but not on 50 mg/L; high resistant (HR), slow growth on 50 g/L; very high resistant (VHR), rapid growth on 50 mg/L. Insensitive (IS), rapid growth with MDPC at 2.5 mg/L or with NPC at 0.5 mg/L; Sensitive (S), no growth with MDPC at 2.5 mg/L or NPC at 0.5 mg/L. C-, not determined. 38 codon 198 for glutamic acid (GAG) in the S strain to codons for glycine (GGG) and lysine (AAG), respectively (Table 1). Characterization of beta-tubulin DNA fragments from other fungal species. Although PCR amplification of genomic DNA from other fungal species with the 22-mer primers was possible, directional cloning of the DNA target sequence was inefficient. Restriction site analysis (data not shown) revealed that the BamHI site, situated immediately downstream from,primer A in amplified beta-tubulin DNA from strains of V. inaequalis, was not conserved in the beta- tubulin DNA of fungi such as A. nidulans (22), E. graminis (32), and N. crassa (26). Directional cloning of PCR products generated with primers C and D was more efficient since both primers contained restriction sites. Specific 436-bp DNA fragments were amplified from strains of V. pirina, M. fructicola, and S. homoeocarpa. All of the cloned DNA fragments showed extensive sequence similarity with the beta-tubulin gene from V. inaequalis. A comparison of the deduced amino acid sequence of beta-tubulin of V. inaequalis with all other fungal species revealed homologies of higher than 96%. In the strains with resistance to benomyl, single point mutations at codon 198, converting glutamic acid to lysine or alanine, or at codon 200, converting phenylalanine to tyrosine were found (Table 1). Agarose gel electrophoresis showed that a specific, slightly larger DNA fragment was amplified from strains of Penicillium species than from strains of the other fungi. 39 Nucleotide sequence analysis of the beta-tubulin DNA fragments cloned from the Penicillium strains showed that an additional 51 bp of sequence were present in the PCR amplified DNA fragments. The extra sequence separated codons 205 and 206 and contained typical fungal intron splice signals (26). The position of this intron was identical to the position of IVS 6 found in the BenA gene of A. nidulans (22). Analysis of the DNA from benomyl- resistant and sensitive strains of Penicillium showed that point mutations were present in codons 198 or 200 from benomyl-resistant strains as previously found in benomyl- resistant strains of V. inaequalis. A new mutation in codon 198 was detected in P. expansum strain EX-301 with a HR phenotype. The codon for glutamic acid in the benomyl- sensitive strain was replaced in strain EX-301 with a codon for valine (Table 1). Field and laboratory-induced mutations in the beta-tubulin gene. A more restricted spectrum of point mutations was found in the beta-tubulin gene from field strains than from laboratory-induced mutants. Five mutations in two codons were found in the beta-tubulin genes of 24 benomyl-resistant field strains (Table 1), versus 14 mutations in nine codons in the beta-tubulin genes of 14 laboratory-induced mutants (Table 2)(6, 13, 14, 26, 35). 40 TABLE 2. Deduced amino acid substitutions in the beta-tubulins of laboratory-induced mutants with resistance to benzimidazole fungicides Amino acid Organism Substitution Position reference Aspergillus nidulans His to Tyr 6 14 His to Leu 6a 13 Tyr to Asn 50b B.R. Oakley Tyr to Ser SOb BAR. Oakley Gln to Lys 134b B.R. Oakley Ala to Val 165C 13 Glu to Asp 198 14 Glu to Gln 198 14 Glu to Lys 198d 14 Phe to Tyr 200d 14 Met to Leu 257b B.R. Oakley Neurospora crassa Phe to Tyr 167 26 Glu to Gly 198d 6 Saccharomyces cerevisiae Arg to His 241 35 aSubstitution confers resistance to benomyl and carbendazim, and tubulin of mutant exhibited decreased affinity for carbendazim (2). bPoint mutations found in heat—sensitive mutants (B.R. Oakley, personal communications). cSubstitution confers resistance to thiabendazole, supersensitivity to benomyl and carbendazim, and tubulin of mutant exhibited increased affinity for carbendazim (2). d Mutations that were found in both laboratory—induced mutants and field strains. DISCUSSION In previous studies involving V. inaequalis and V. pirina, strains were classified as S, LR, MR, HR, or VHR based on their differential growth response on PDA and diethofencarb (11, 16, 29, 30, 31, 34). Among 24 benomyl-resistant field strains, all except three strains with low resistance had mutations in codon 198 or codon 200 of the beta-tubulin gene. The results of this study indicate that a change in codon 198 or codon 200 of the beta-tubulin of V. inaequalis and V. pirina and of several other plant pathogens confers resistance to benomyl and implies a key role of the amino acids at these positions in the action of benzimidazole fungicides. In addition, each phenotypic class of V. inaequalis, with the exception of the LR class, was associated with a unique amino acid substitution at position 198 or 200. The same association between unique amino acid substitutions and benomyl-resistance levels was observed in field strains of V. pirina, M. fructicola, S. homoeocarpa, and six species of Penicillium. Results from Southern analysis performed at moderate stringency suggested that V. inaequalis contains only one copy of the beta-tubulin gene. This result in conjunction with previous genetic data suggesting that only one benomyl- 41 42 resistance gene exists in V. inaequalis support the assumption that the observed mutations in codons 198 and 200 are directly responsible for benomyl-resistance in this plant pathogen. We did not investigate whether additional beta-tubulin sequences were present in the other fungi by Southern analysis with their homologous probes. Our data were consistent with the hypothesis that certain mutations in the beta-tubulin gene confer sensitivity to diethofencarb as well as resistance to benomyl (6, 11, 30). Only those mutations in codon 198, converting the codon for glutamic acid in sensitive strains to alanine in resistant strains of six fungal species with the VHR phenotype, or to glycine in one MR- strain of V. inaequalis, were associated with a change in phenotype from insensitivity to sensitivity to diethofencarb. Other substitutions in codon 198 did not automatically confer sensitivity to all N-phenylcarbamate fungicides. HR strains, with mutations changing codon 198 for glutamic acid to codons for lysine or valine, exhibited sensitivity to MDPC but insensitivity to diethofencarb. In addition, mutations in codon 200 from a phenylalanine codon to tyrosine codon, were never associated with an altered sensitivity to diethofencarb. In transformation experiments with N. crassa, a mutation of codon 198 from a glutamic acid codon to a glycine codon conferred resistance to benomyl and sensitivity to diethofencarb (6). This mutation was also found in benomyl-resistant and diethofencarb-sensitive strain I-26 of V. inaequalis (Table 1). 43 The characterization of laboratory-induced mutants of model organisms such as N. crassa and A. nidulans has been very important for establishing the mode-of-action of the benzimidazole fungicides (3, 13, 14, 26). However, their value in predicting whether a particular mutation will become a problem in the field remains unknown. The mutations in the beta-tubulin gene of benomyl-resistant field strains of several plant pathogenic fungi were limited to two codon positions (Tables 1 and 2). A possible reason for the reduced variation in mutations found among field strains as compared to laboratory-induced mutants is that mutations in other codons might interfere with the fitness of mutants and impose a selective disadvantage on these mutants under field conditions. Benomyl-resistant strains of A. nidulans with mutations in codons 50, 134, or 257 were also temperature-sensitive (B.R. Oakley, personal communication) (Table 2), and such strains would be expected to have a reduced fitness. Results from greenhouse and field studies indicate that many benomyl-resistant field strains exhibited a high level of fitness as evidenced by their persistence in populations (19, 23). Some field strains of V. inaequalis collected from an orchard in Michigan more than 10 yr after benomyl application was discontinued contained a mutation in codon 198 from glutamic acid to alanine and exhibited very high resistance to benomyl in laboratory tests (18). However, a comparison of the deduced amino acid sequences from beta-tubulin genes of 44 many organisms revealed a highly conserved glutamic acid in position 198 suggesting that there is considerable constraint on this residue (14). The observation that mutants with non-conservative amino acid substitutions at position 198, from glutamic acid to lysine, glycine, or alanine, are common in the field was therefore surprising. The appearance of field mutants with a substitution of phenylalanine by tyrosine in position 200 was less surprising. A comparison of the amino acids in position 200 showed that considerable variation exists among different organisms and suggests a reduced constraint on this position. Provided that mutations in other positions than 198 or 200 of the beta-tubulin gene interfere with the fitness of mutants, it will be important to use field mutations in the genetic engineering of strains of fungi that are in use in biocontrol (27). Differential affinity of fungal tubulin to both N- phenylcarbamates and carbendazim, the toxic principle of benomyl, is likely the basis for the variation in resistance among the benomyl-resistant strains examined in this study. Reduced binding of carbendazim by crude extracts of tubulin was reported for laboratory-induced strains of A. nidulans (3), field strains of V. nashicola (9), and thiabendazole- resistant strains of the nematode Haemonchus contortus (20). The beta-tubulin genes of the A. nidulans strains used in binding studies by Davidse (3) contained unique codon substitutions that conferred resistance or supersensitivity 45 to benomyl (13). In benomyl-resistant strain BEN17, the BenA19 allele had a leucine codon in place of a histidine codon at position 6, and tubulin from this strain exhibited reduced affinity for carbendazim. Replacement of an alanine codon with a valine codon at position 165 in the BenA16 allele of strain BEN14 was associated with both supersensitivity to benomyl and increased affinity of tubulin for carbendazim. Evidence implicating tubulin in the mode of action of N-phenylcarbamates was that germinating spores of VHR strains exposed to diethofencarb, MDPC, or N-phenylformamidoximes exhibited distortion of germ tubes identical to that observed when benomyl-sensitive strains were exposed to carbendazim (17, 25). In addition, crude protein extracts from strains of V. nashicola with different levels of resistance to benomyl exhibited differential binding of N-phenylformamidoximes (10). The beta-tubulin genes of the mutants were not characterized. Such results indicate that amino acids in positions 198 and 200 in beta-tubulin may play a critical important role in the binding of N-phenylcarbamates and carbendazim. Substitution of glutamic acid at position 198 by glycine or alanine in benomyl-resistant strains could dramatically increase the affinity for both diethofencarb and MDPC, while substitution of glutamic acid by lysine or valine could increase the affinity for MDPC but not for diethofencarb. A similar model for differential binding of carbendazim and diethofencarb to beta-tubulin of benomyl-sensitive and 46 benomyl-resistant strains was proposed by Fujimura et al. (6). Structural analysis of carbendazim, N- phenylcarbamates, and N-phenylformamidoximes compounds with Alchemy II software (Tripos Associates, St. Louis, MO) revealed nearly superimposable shapes for the molecules (Koenraadt, unpublished). However, structural analysis of the beta-tubulin protein is necessary to substantiate the hypothesis that the N-phenylcarbamates fungicides bind to the altered carbendazim binding site, but such a study has not been possible because of the intractable problem at crystallizing tubulin for x-ray diffraction studies (1). The molecular basis for benomyl-resistance in strains with the LR phenotype was not elucidated. Classic genetic analysis provided evidence that this trait was heritable and controlled by the same gene that conferred MR, HR, and VHR phenotypes to benomyl in V. inaequalis and V. pirina (29, 31, 34). Only a portion of the beta-tubulin gene of LR strain MINNS 118 was sequenced and a mutation might be present in a portion of the gene that was not sequenced. It may also be possible that low resistance to benomyl was due to altered expression of the beta-tubulin gene. Characterization of a laboratory-induced benomyl-resistant strain of Epichloe typhina revealed a rearrangement in the 5'-flanking region of the beta-tubulin gene and no alternations in the deduced amino acid sequence were found (2). Sequence variation in the 5'-flanking region was observed in LR strain MINNS 118 (Koenraadt, unpublished 47 results), but whether this variation altered the expression of the beta-tubulin gene and was responsible for the LR phenotype was not investigated. LITERATURE CITED 1. Burns, R.G. 1991. Alpha-, beta-, and gamma-tubulins: sequence comparisons and structural constraints. Cell Motil. Cytoskel. 20:181-189. 2. Byrd, A.D., Schardl, C.L., Songlin, P.J., Mogen, K.L., and Siegel, M.R. 1990. The beta-tubulin gene of Epichloe typhina from perennial ryegrass (Lolium perenne). Curr. Genet. 18:347-354. 3. Davidse, L.C., and Flach, W. 1977. 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Further studies on the inheritance of benomyl-resitance in Venturia pirina isolates from pear orchards in Israel. Plant Pathol. 35:310-313. 51 32. Sherwood, J.E., and Somerville, S.C. 1990. Sequence of Erysipbe graminis f.sp. hordei gene encoding beta-tubulin. Nucl. Acids Res. 18:1052. 33. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophores1s. J. Molec. Biol. 98:503-517. 34. Stanis, V.F., and Jones, A.L. 1984. Genetics of benomyl resistance in Venturia inaequalis from North and South America, Europe and New Zealand. Can. J. Plant Pathol. 6:283-290. 35. Thomas, J.H., Neff, N.F., and Botstein, D. 1985. Isolation and characterization of mutations in the beta- tubulin gene of Saccharomyces cerevisiae. Genetics 112:715- 734. 36. Zhang, H., Scholl, R., Browse, J., and Somerville, C. 1988. Double stranded DNA sequencing as a choice for DNA sequencing. Nucl. Acid Res. 16:1220. PART II THE USE OF ALLELE-SPECIFIC OLIGONUCLEOTIDE PROBES TO CHARACTERIZE RESISTANCE TO BENOMYL IN FIELD STRAINS OF VENTURIA INAEQUALIS ABSTRACT A procedure was developed for detecting point mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis, using the polymerase chain reaction (PCR) in combination with allele-specific oligonucleotide (ASO) analysis. PCR was used to amplify a specific 1,188-bp DNA sequence of the beta-tubulin gene in DNA extracts from axenically grown mycelium or individual apple scab lesions. The amplified DNA sequence was then probed with 18-mer end- labeled oligonucleotides specific for sensitive or for three benomyl-resistant phenotypes in strains of V. inaequalis. The point mutations, converting codon 198 from glutamic acid in the sensitive strain to lysine or alanine, were detected by ASO analysis in highly resistant and very highly resistant strains, respectively. A point mutation, converting codon 200 for phenylalanine in sensitive strains to tyrosine, was detected by ASO analysis in all medium resistant strains but one from Chile, isolate Chile 24B. Sequence analysis revealed that an alternate codon for tyrosine was present in the Chilean isolate. Therefore, the mutation in isolate Chile 248 was not detected by any of the four ASO probes used in this study. ASO analysis was an useful tool for detecting and characterizing benomyl- 52 53 resistant strains of V. inaequalis and could be expanded to other plant pathogenic fungi. INTRODUCTION Benzimidazole compounds are widely used as fungicides and anthelmintics, and are also promising antineoplastic agents (3). Inherent to the site-specific mode of action of benzimidazole compounds is the potential for developing resistance in target organisms. Point mutations in the beta-tubulin gene have been shown to confer resistance to the benzimidazole fungicide benomyl (5, 8, 11, 13, 20). Benomyl resistant strains of Venturia inaequalis (Cke.) Wint. have been isolated in many apple growing regions worldwide. In addition to sensitive (S) strains, four classes of resistant strains (low, LR; medium, MR; high, HR; and very high resistant, VHR) were established based on the growth response of the strains on media amended with benomyl (9, 14, 17). Moreover, HR and VHR strains were differentiated from each other based on sensitivity of VHR strains to diethofencarb (7, 16). Studies on the genetic basis of benomyl resistance indicated that resistance to benomyl in Venturia was determined by a single gene (9, 14, 15, 17). Sequence analysis of the beta-tubulin DNA of field strains of V. inaequalis revealed that point mutations in codons 198 or 200 were associated with differential resistance to benomyl. Conversion of codon 200, from 54 55 phenylalanine in a sensitive strain to tyrosine, was associated with medium resistance to benomyl. Conversion of codon 198, from glutamic acid to lysine or alanine, was associated with high or very high resistance to benomyl, respectively (11). DNA probes have been developed to detect bacteria with resistance to copper or streptomycin (2, 6, 12). Unlike detection of benomyl resistance, in which point mutations are responsible for resistance, the detection of copper or streptomycin resistance is based on the presence of genes that are absent in sensitive strains. Genetic disorders caused by point mutations in DNA of humans are now detected by the combined use of the polymerase chain reaction (PCR)(4) and allele-specific oligonucleotide (ASO) analysis (1, 19). PCR is used to amplify the appropriate DNA sequence and then ASO analysis is used to detect the single base pair mutations. The objective of this study was to develop ASO analysis for the rapid detection of point mutations that confer resistance to benomyl in field strains of V. inaequalis. ASO analysis was used to establish the relatedness of the allelic mutations in a collection of field strains isolated in different regions of the world. Individual apple scab lesions were subjected to ASO analysis to characterize allelic mutations in strains from an orchard in which benomyl had not been applied for more than 10 years. MATERIALS AND METHODS Fungal strains. The single-spore isolates of V. inaequalis used in this study were from a large collection of field strains previously characterized in studies on the inheritance of resistance to benomyl and of negatively- correlated cross resistance to diethofencarb (7, 17). The strains originated in different geographic regions of the world and exhibited widely varying levels of resistance to benomyl. Strains IL-2, IL-3, IL-6, IL-7, and IL-8 of V. pirina Aderhold were provided by E. Shabi, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. Each strain was designated as sensitive (S), low resistance (LR), medium resistance (MR), high resistance (HR), or very high resistance (VHR) to benomyl based on its growth response on media amended with benomyl or diethofencarb (6, 9, 14, 15, 16). DNA isolation and polymerase chain reaction. Total DNA was isolated from mycelium from cultures grown in potato dextrose broth (PDB) or on potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI) as previously described (11), except that the mycelium was macerated with disposable pellet pestles (Kontes, Morton Grove, IL) in microcentrifuge tubes. The isolated DNA was then used as template in the 56 57 PCR. Two 22-mer oligonucleotides, 5'-CAAACCATCTCTGGCGAACACACG and 5'-TGGAGGACATCTTAAGACCACG, which were identical in sequence to codons 22 to 28 and complementary in sequence to codons 359 to 365 of the beta- tubulin gene of V. inaequalis, respectively, were used as PCR primers to amplify a 1,188—bp DNA sequence. PCR was performed and amplification products were analyzed by agarose gel electrophoresis as described previously (11). Allele-specific oligonucleotide probes. Four ASO probes for detecting allelic mutations in the beta-tubulin gene of V. inaequalis were synthesized in the Macromolecular Structure Facility, Department of Biochemistry, Michigan State University, East Lansing. The 18-mer oligonucleotides were designated as ASOS'LR, ASOMR, ASOHR, and ASOVHR probes based on the specificity of each probe. Each ASO probe included the sequences of codons 198 and 200 (Fig. 1A) because mutations in these codons were associated with resistance to benomyl in field strains of V. inaequalis (11). A80 probes were end-labeled with gamma-32P-ATP (Du Pont, Boston, MA) using T4 polynucleotide kinase (Promega Corporation, Madison, WI) to a minimum specific activity of 2 x 109 dpm/pmol. Allele-specific oligonucleotide analysis. PCR amplified beta-tubulin DNA (25 ng/sample) was denatured in 0.25 N NaOH for 10 min and then applied to a nylon membrane (GeneScreen- Plus, Du Pont, Boston, MA) in a dot blot manifold. The dot blots were incubated in prehybridization solution (1 M NaCl, 58 A; Allele-specific oligonucleotide probes for Venturia inaequalis S-LR 198 200 A80 50 TOT GAC GAG ACA TTC TG 3’ ' MR V ASO C GAG ACA TAC TGC ATT GA HR V ASO C TCT GAC AAG ACA TTC TG VHR V ASO C TCT GAC GCG ACA TI'C TG _ Number of 8. Sequence for codons 195 to 203 of the beta-tubulin gene mismatches Venturia inaequalis AAC TCT GAC GAG ACA TTC TGC ATT GAC O V. pirina AAI TCG_ GAC GAG AC9 TTC TGC ATT GAC 3 Moniliniafructicola AAC TCT GA: GAA ACQ TTC TGT ATC GAT 3 Penicillium aurantiogrzls‘ezun CAC TCQ GAC GAG ACQ TTC TGT ATC GAT 2 P. digitatum CAC TCQ GAC GAG ACI TTC TGT ATC GAT 2 P. expansum CAC TCQ GAC GAG AC]: TTC TGT ATC GAT 2 P. italicum CAC TCQ GAC GAG ACI TTC TGT ATC GAT 2 P. puberulum CAC TCQ GAC GAG ACQ TTC TGT ATC GAT 2 P. viridicatum CAC TC(_3_ GAC GAG ACQ TI’C TGT ATC GAT 2 Sclerotinia homoeocarpa AAC TCT GAC GAG ACC_3 TTC TGT ATC GAT _A Fig 1. A, Sequence of allele-specific oligonucleotide (ASO) probes for Venturia inaequalis. The A80 probes for medium (MR), high (HR), and very high resistance (VHR) to benomyl differ from the beta-tubulin DNA for sensitive (S) and low resistance (LR) strains by one nucleotide (arrows). B, Sequence of codons 195 to 203 of the beta-tubulin gene from benomyl-sensitive field strains of 10 plant pathogenic fungi. Under conditions of high stringency, the ASOS-LR probe will be removed from its complementary anti-sense strand sequence from fungi other than V. inaequalis due to one to three mismatches (underlined) in the third position of codons. 59 50 mM Tris-HCl, pH 7.5, 10% dextran sulfate, 1% SDS, 0.2% Ficoll (M.W. 400,000), 0.2% polyvinylpyrrolidone (M.W.40,000), 0.2% bovine serum albumin, 0.1% sodium pyrophosphate, and 0.25 mg/mL denatured salmon sperm DNA) for 2 h at 65 C according to the manufacturer's procedure. An end-labeled ASO probe was then added to the prehybridization solution and incubated at 37 C for at least 4 h. The blots were washed three times for 15 min each in 2x SSC buffer (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8) at room temperature. A high stringency wash, three times for 2 min each in 2x SSC buffer, was then used to remove ASO probe with a single base pair mismatch from the blots. The optimum temperature for the high stringency wash with each probe was determined empirically. The dot blots were exposed to X-ray film for 0.5-2 day at - 70 C. Direct characterization of alleles in apple scab lesions. Leaves and fruits with scab lesions were collected in an apple orchard at the Botany and Plant Pathology Farm on the campus of Michigan State University, East Lansing. Monitoring of the apple scab population in 1976 revealed the existence of a S and VHR population in this orchard (Jones, unpublished). The resistance level of spores from individual lesions was determined by plating conidia from each lesion on PDA amended with 0, 0.5, 5, or 50 mg/L benomyl or 1 mg/L diethofencarb (7, 11, 17). Lesions were cut from leaves or fruits with a scalpel and individually 60 transferred to microcentrifuge tubes. Isolation of DNA from the lesions, amplification of the target sequence by the PCR, and A50 analysis were done as described above. DNA isolated from healthy leaves and fruits was included as a control to determine whether there was significant PCR amplification of other DNA sequences. RESULTS Allele-specific oligonucleotide analysis. DNA isolated from as little as 10 mg fresh weight of mycelium from broth or agar plate cultures was sufficient for the amplification of the 1,188-bp target sequence of the beta-tubulin gene using PCR. Amplified DNA was not detected when sample DNA was omitted from the PCR reaction mixture. Each of the four ASO probes hybridized to the PCR amplified beta-tubulin DNA of all strains of V. inaequalis after washing the dot blots at 50 C (Fig. 2A). After washing at 64 C, the ASOS'LR probe hybridized only with the PCR amplified beta-tubulin DNA of strains with S or LR phenotype, and not to DNA of strains with the MR, HR, or VHR phenotype (Fig. 25). The ASOMR, ASOHR, and AsoVHR probes exhibited allele-specific hybridization after washing at 61, 61, and 63 C, respectively (Fig. 2C-E). Amplified beta-tubulin DNA from strain Chile 24B of V. inaequalis with medium resistance to benomyl did not hybridize with the ASOMR probe nor with the ASOS'LR, ASOHR, or ASOVHR probes under conditions of high stringency. Amplified beta-tubulin DNA from strain CHILE 24B was sequenced and contained a mutation in codon 200 of the beta- tubulin gene. The codon for phenylalanine (TTC) found in 61 62 TABLE 1. Benomyl resistance phenotypes of field strains of Venturia inaequalis from.th nbc ...SAK..QAY..S..A ........ A ...... C.P ...... VA. M...RM..R..0... MS ................. K..V.D ..... C. elega‘s hen-1 ...SAK..QAY..L..A ........ A ...... C.P.H ..... VA. M...RM..R..D.. M.... ........ ..K..V 0.... C. elegans tub-1 ...SAK.TQAY..L..A ........ A ...... C.P.H ..... VA. M...RM..R..DE..L .................. K..V.D ..... C. elegas tree-1 . .TSRSIKnY. I..P .. C..A.. ...C P. H.. ..AA ..... RM ..... DE..L.I ......... 0 ...... K..V.0 ..... P. falciparun ....... smv..L ........... A....C...P.H ..... AC. M.. ..RM T. ..0E..L ......... Y ..... H. TKSSV. D...K. 0. nelmogaster ..... ...snov .L.. ...........A......CPH ..VA ..... RM.....DE..L.I ...... ..... K..V. 0... G. wax ....... SNY. SL I ....... HUAR ..C.....APH ...A. .M.. ....MT ..00..I .................. KSSV. 0...T. A. thaliam ....... SIJYISL. .. ......LJ A....C. A. P. H. ....A..M.. .M..T...DE.IL .................. KSSV.D...T. H. sapiens ....... SCDY..L ........... A ...... C.P. H ..... VA. V. . .RM.....0...L ........... K..V.D ..... 370 380 390 4(1) 410 420 430 440 Ref V. inaeqalis LKMSSTFVGISTSICELFKRVEQHMFRRKAFLMTCEGDBEFTEAEWLVSEYmYCEASVSEEEEWEEAPLEGEE 29 N. crassa AIE .......... ..... ........0AGVDEEEEEYEEEAPLECEE 41 A. nidlla‘s ben1 ..... 1.... ...................................................... D.EISNDELEEAYEEINPLEG 36 H. contortus tLbC ...AA..I....A ....... ISE ........................................... EATPDDIQLDAEGLEEAYPEE 14 C. elegans ban-1 ....A..I....A.......ISE ..................................... . ..... EATAEEDCELDGTDGME 15 C. elegas tlb-‘l ...AA ....... A ....... ISE ................................... I ....... EATAEJIMDGYAEEAIITYESEQ‘IS C. elega's nee-1 ....A..I....A ....... ISE ........................................... EAAADEDAAEAFDCE 15 P. falciparun ...AV.......A...M....S ............................................ DATAEEEEFEEEECDVEA 8 0. "elapgastor . AIAISE .................... . ...... . ...... ...EATPDEDAEFEEEEAEVDEN 37 G. mx .8 ..... M ........ M.R..SE...V..K .................... VRA ...... A ...... DATAVDDHEDEDEDEAMAA 16 A. thaliem I..A ............ M.R..SE ........................................... DATPDEEDEYDEEEEQVYES 40 H. smiers ....A..I....A ....... IE ....................................... ....DATAEEEEFEEEAEEEVA B 103 microheterogeneity among beta-tubulin isotypes also could interfere with the affinity of tubulin for benzimidazole compounds. Therefore, we hypothesized that microheterogeneity among beta-tubulin isotypes is the basis of selective toxicity of compounds of benzimidazoles and phenylcarbamate chemistry, as well as colchicine. Several studies provide evidence to support our hypothesis. In the nematode Caenorhabditis elegans benzimidazole mutants carried a deletion of the ben-l gene, one of the three beta- tubulin genes of this organism. Therefore, the product of the ben-l beta-tubulin gene, but not the products of the tub-1 and mec-7 beta-tubulin genes, are sensitive to benzimidazoles (9). The microheterogeneity among the hen-1, tub-1, and mec-7 beta-tubulin isotypes from C. elegans is relatively limited (Fig. 2). Six unique amino acid changes at positions 56, 218, 231, 278, 328, and 331 within the region before amino acid 430 of ben-l were identified that correlated with benzimidazole sensitivity (9). Also, a binding study showed the differential affinity of benzimidazoles to tubulin from mammalian brain and parasitic nematodes (44). A conclusion of this study was that the selective toxicity of benzimidazoles was directly related to the differential affinity of host and helminth tubulins for these compounds. A review of binding studies of colchicine to tubulin from several different species showed the existence of differential affinity of tubulin to this compound (2). A binding study of colchicine to beta-tubulin 104 isotypes, purified from bovine brain and separated by immunoaffinity chromatography, showed that two beta-tubulin isotypes had a significantly different affinity for colchicine (1). Provided that microheterogeneity among beta-tubulins is the basis of selective toxicity of benzimidazoles, phenylcarbamates, and colchicine, it is important to assign amino acid residues that are critical for the binding of these compounds. A considerable number of amino acid substitutions exist among beta-tubulin isotypes, hindering a proper evaluation of their role in the binding of these compounds (Fig. 2). Characterization of beta-tubulin genes from benzimidazole-resistant mutants suggests that substitution of amino acid residues at positions 50, 134, 165, 167, 198, 200, 241, 257, or 350 are critical for the binding of those compounds (13, 23, 24, 29, 41, 49, Oakley, personal communication). For instance, Jung and Oakley conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazoles compounds (23). Therefore, we compared the amino acid residues in beta-tubulin isotypes from several organisms at these positions. A comparison of amino acid residues at putative critical positions in beta-tubulins revealed that considerable, and to a certain extent phylum-specific, variation exists among organisms at positions 165 and 200, and to a lesser extent at 257 and 350 (Table 1) (23). The variation in residues at 105 TABLE 1. Comparison of the deduced amino acid residues in beta-tubulin at positions that might be critical for resistance to benzimidazoles and related compounds. Amino acids in positions Species (Isotype) 6 50 134 165 167 198 200 241 257 350 Ref V. inaequalisa His Tyr Gln Ala Phe Glu Phe Arg Met Gln 27 E. graminis - - - - - - - - - - 47 N. crassa - - - - - - - - - - 41 A. nidulans (benA) - - - - - - - - - - 36 S. commune (tub-2) - - - Cys - - - - Leu Lys 42 A. klebsiana - - - Cys Tyr - Met - Leu Lys 3 P. carinii - — - - - - - - - — 10 H. contortus (tub8-9) - - - - - - - - - Lys 14 H. contortus (tub12-16)- - - Ser - - - - - Lys 14 H. contortus(tub12-164)— — - Ser — - - - - Lys 14 C. elegans (ben-I) - - - Ser - - - - - Lys 15 C. elegans (tub-1) - — - Ser — - Tyr - - Lys 9 C. elegans (mec-7) - - - Asn - Ser - - - Lys 45 L. mexicana Ser - — Metb - - Met — Leu Lys 11 B. pahangi - - - Ser - - - - - Lys 17 P. falciparum — Phe - Glu - - Gln - Leu Lys 8 T. brucei Cys — - Metb - - Met - Leu Lys 26 D. melanogaster(beta-1)- - — Asn Tyr - Tyr - - Lys 37 G. max (S-beta-I) - - - Leu - - Met - Leu Lys 16 A. thaliana (beta-1) - — - Leu - - Met - Leu Lys 40 Z. mays beta-1 - - - Leu - - Met - Leu Lys 19 G. gallus - - - Asn - - Tyr - — Lys 50 H. sapiens (beta-2) - - - Asn - - Tyr - - Lys 33 aAmino acids residues in the beta-tubulin of V. inaequalis are shown at top. Only amino acids that differ from V. inaequalis are shown, while similar amino acids are indicated by dashes. bAmino acid residue deletion is present at position 165 of the beta- tubulin. Motif MMATF163'167 is reduced to MMTF164-167 in the beta-tubulin of V. inaequalis in L. mexicana and T. brucei. 106 positions 6, 50, 167, and 198 is very limited, while no variation exists among residues in position 134 and 241 in our sample of beta-tubulins (Table 1). No or limited variation suggests that structural constraints prevent variation in amino acid residues in these positions. A tyrosine residue at position 200 is common for mammalian beta-tubulins (23). The fact that the substitution 200Phe to Tyr confers resistance to benomyl in several fungi suggests that tyrosine at this position in mammalian beta- tubulin might be an important factor for insensitivity to this compound (23, 27). A methionine residue is common at position 200 among beta-tubulins of plants (Table 1). For instance, in all of the nine beta-tubulin isotypes of A. thaliana, a methionine is present at position 200 as part of the plant-specific CMVL199'203 beta-tubulin motif (48). Arguably, this residue or motif might be responsible for the selective toxicity of herbicides of phenylcarbamate chemistry. A comparison of the beta-tubulin isotypes encoded by the ben-l and tub-1 genes suggest that the presence of 200Tyr in tub-l rather than the previous mentioned residues in positions 56, 218, 231, 278, 328, and 331 is responsible for resistance in benzimidazole-resistant mutants in which the ben-l gene is deleted (Fig. 2, Table 88er in the mec-7 beta- 1). In addition, the presence of 19 tubulin gene might explain for the insensitivity of this beta-tubulin isotype since mutations in this codon conferred resistance to benomyl (24, 29) in several fungi. In this 107 particular case, the variation can not be classified as phylum-specific variation. The presence of phylum-specific variation among critical residues in beta-tubulins can be exploited to design new compounds that interfere with tubulin assembly. However, it is likely that resistance will develop under a continuous selection pressure of these new compounds as previously experienced with benzimidazole compounds such as benomyl and thiabendazole. The key question is whether mutations that will confer resistance to the new compounds interfere with the fitness of the organism under natural conditions. In V. inaequalis, VHR field strains with substitutions in position 198 appear to have a comparable fitness with benomyl- sensitive strains (29), despite the fact that constraints are thought to exist at this position (Table 2) (24, 29). In addition, the existence of extensive phylum-specific variation at positions 165 and 200 implies that a reduced constraint also exists at these critical residues. 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Nakata, A., Sano, S., Hashimoto, S., Hayakawa, K., Nishikawa, H., and Yasuda, Y. 1987. Negatively correlated cross-resistance to N-phenylformamidoximes in benzimidazole- resistant phytopathogenic fungi. Ann. Phytopathol. Soc. Jpn. 53:659-662. 40. Oppenheimer, D.G., Haas, N., Silflow, C.D., and Snustad, D.P. 1988. The beta-tubulin gene family of Arabidopsis thaliana:preferred accumulation of the beta-1 transcript in roots. Gene 63:87-102. 41. Orbach, M.J., Porro, E.B., and Yanofsky, C. 1986. Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol. Cell. Biol. 6:2452-2461. 112 42. Russo, P., Juuti, J.T., and Raudaskoski, M. 1991. Cloning, nucleotide sequence and expression of the beta- tubulin gene from the homobasidiomycete Schizophyllum commune. Unpublished. 43. Russell, G.J., and Lacey, E. 1992. Differential stability of the benzimidazole (BZ)-tubulin complex in B2- resistant and BZ-susceptible isolates of Haemonchus contortus and Trichostrongylus colubriformis. Int. J. Parasitol. 22:399-402. 44. Russell, G.J., Gill, J.H., and Lacey, E. 1992. Binding of [ H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics. Biochem. Pharmacol. 43:1095-1100. 45. Savage, C., Hamelin, M., Culotti, J.G., Coulson, A., Albertson, D., and Chalfie, M. 1989. Mec-7 is a beta-tubulin gene required for the production of lS-protofilament microtubules in Caenorhabditis elegans. Genes Dev. 3:870- 881. 46. Shabi, E., Koenraadt, H., and Dekker, J. 1987. Negatively correlated cross-resistance to phenylcarbamate fungicides in benomyl-resistant Venturia inaequalis and V. pirina. Neth. J. Plant Pathol. 93:33-41. 47. Sherwood, J.E., and Somerville, S.C. 1990. Sequence of Erysiphe graminis f.sp. hordei gene encoding beta-tubulin. Nucl. Acids Res. 18:1052. 48. Snustad, D.P., Haas, N.A., Kopczak, S.D., and Silflow, C.D. 1992. The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes. Plant Cell 4:549-556. 49. Thomas, J.H., Neff, N.F., and Botstein, D. 1985. Isolation and characterization of mutations in the beta- tubulin gene of Saccharomyces cerevisiae. Genetics 112:715- 734 50. Valenzuela, P., Quiroga, M., Zaldivar, JU., Rutter, W.J., Kirschner, D., and Cleveland, D.W. 1981. Nucleotide and corresponding amino acid sequence encoded by and beta- tubulin mRNAs. Nature 289:650-655. 51. Wilson, L. 1970. Properties of colchicine binding protein from chicken embryo brain. Interactions with vinca alkaloids and podophyllotoxin. Biochem. 9:4999-5007. APPENDIX B RECOMMENDATIONS RECOMMENDATIONS RAPID DETECTION OF POINT MUTATIONS A number of techniques are available to characterize point mutations in DNA. Single base pair mutations in the beta-tubulin gene from several fungi were detected by dideoxynucleotide sequence analysis (3). However, this method is rather laborious and time-consuming. Allele- specific oligonucleotide (ASO) probes (18-mers) were used to determine point mutations in PCR-amplified beta-tubulin DNA of Venturia inaequalis (2). Sequence analyses showed that alternate codons were encoding identical amino acid residues around positions 198 and 200 of beta-tubulin from different plant pathogenic fungi. Therefore, species-specific ASO probes are required to detect point mutations in different fungi (2). Selective amplification of DNA was used to discriminate wild-type from mutant strains with point mutations in codons 74 or 215 of the gene encoding for reverse transcriptase of the human immunodeficiency virus type-1 (6). In this study, different primer pairs, 17 and 21-mer oligonucleotides with a variable nucleotide at their 3'-ends, were used to amplify DNA. Agarose gel electrophoresis of the PCR products was used to show whether a point mutation was present in the target DNA (6). An advantage of selective amplification over ASO analysis is that even less time is required to 113 114 determine whether a mutation is present in target DNA. In addition, the method of selective amplification does not require the use of radiolabelled DNA. In theory, amplification with 10-mer primers reduces the chance that alternate codons interfere with the amplification of the target DNA. However, at random amplification of non-target sequences increases when shorter primers are used (7). Therefore, it will be of interest to investigate whether 10-mers can be used as nested primers in amplification reactions. A short number of amplification cycles under relatively stringent annealing conditions can be used to amplify beta-tubulin DNA with generic beta- tubulin primers as described in a previous study. Then, the amplification could be continued at lower stringency to allow allele-specific amplification with the 10-mer primers. Preliminary experiments, in which plasmid DNA was used as template, showed that 10-mer oligonucleotides with a variable nucleotide at their 3'-ends can be used for the selective amplification of beta-tubulin DNA (Koenraadt, unpublished). However it remains to be investigated whether selective amplification with 10-mer primers is feasible in reactions involving genomic DNA. IS MICROHETEROGENEITY THE BASIS FOR SELECTIVITY 0F BENZIMIDAZOLES AND PHENYLCARBAMATES? Microheterogeneity among beta-tubulins is likely the basis for the selective toxicity of benzimidazoles and phenylcarbamates (1). Site-directed mutagenesis in the 115 beta-tubulin gene from Neurospora crassa is necessary to prove whether this hypothesis has validity. In plants, methionine is in general present at position 200 as part of the plant-specific CMVL199'203 motif (1). Also, the alteration of codon 200 for phenylalanine to tyrosine confers resistance to benomyl (2, 3). Provided that methionine at position 200 or possible more residues of the motif are responsible for the selectivity of phenylcarbamate herbicides, alteration of one or more codons at positions 199-203 should increase the sensitivity to these herbicides. Transformation experiments with spheroplasts of N. crassa could be used to determine whether transformants in which the beta-tubulin constructs are expressed confer sensitivity to phenylcarbamate herbicides. Experimental evidence to support the hypothesis will be obtained when fungal transformants appear to be sensitive to phenylcarbamate herbicides. TRANSFORMATION OF ASPERGILLUS NIDULANS In Neurospora crassa, DNA usually integrates randomly into the genome upon successful transformation. Gene- replacement, rather than random integration, is the major mode of integration of DNA in transformation experiments with Aspergillus nidulans. Binding and transformation experiments showed that beta-tubulin isotypes have differential affinity for benzimidazole compounds. Therefore, the presence of multiple beta-tubulin isotypes as 116 observed in most higher eukaryotic organisms disguises the sensitivity of one particular beta-tubulin isotype to benzimidazole and phenylcarbamate compounds as reviewed in this thesis (1). A. nidulans contains two beta-tubulin isotypes that are encoded by the benA and tubC genes (5). Deletion of the tubC gene which is only expressed during conidiation does not affect the viability of A. nidulans under laboratory conditions (4). Therefore, the phenomenon of gene replacement in transformation experiments with tubC' strains of A. nidulans can be exploited in gene replacement experiments with benA. In the tubC” background, beta- tubulins encoded by introduced foreign genes could be analyzed in the absence of endogenous beta-tubulin isotypes. LITERATURE CITED 1. Koenraadt, H., and Jones, A.L. 1992. Mutations in codons 198 and 200 of the beta-tubulin gene confer resistance to benomyl and codon 198 also confers sensitivity to diethofencarb. This thesis. 2. Koenraadt, H., and Jones, A.L. 1992. The use of allele- specific oligonucleotide probes to characterize resistance benomyl to benomyl in field strains of Venturia inaequalis. Phytopathology 82 (in press). 3. Koenraadt, H., Somerville, S.C., and Jones, A.L. 1992. Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogens. Phytopathology 82 (in press). 4. May, G.S. 1989. The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. J. Cell Biol. 109:2267-2274. 5. May, G.S., Tsang, M.L.-S. Tsang, Smith, H., Fidel, S., and Morris, N.R. 1987. Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene 55:231-243. 6. St. Clair, M.H., Martin, J.L., Tudor-Williams, G., Bach, M.C., Vavro, C.L., King, H.M., Kellam, P., Kemp, S.D., and Larder, B.A. 1991. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science 253:1557-1559. 7. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535. 117 y... n._>............. HICHIGQN STQTE UNIV LIBRQRIES l (W 1111“ 97L0 I 1 11W W 111 H I 1 3129301055 . .. .. . _....;.i..,\.£....H.. . ... .... ... ... .....t. .. . ... ,.................. .. .... .....N... ....-. .. , .. “.19.. . .. . . ~14 E. L..: . .....zl .....1... .. ........ ...T.... .....i... ...T}... . ....J.... .Y..... ....... _.......1.....,. ..i... ..., . ...}...Q‘. . .. . 3.2... ., . . . . ...I. .._...._.. 7.... .. . ...T... ., ......... . .. 2. ... I 9:. , . s z: t. _;.. 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