Revertant Strains Research Articles

Experimental study of ascending Candida albicans pyelonephritis focusing on the hyphal form and oxidant injury.

To investigate the pathogenicity of the hyphal form of Candida in pyelonephritis a Candida albicans strain, assuming only the yeast form but not the hyphal form when induced by ultraviolet mutagenesis, and a revertant strain from this mutant strain showing bimorphism were compared in a rat experimental model with regard to the incidence of ascending Candida pyelonephritis and the grade of inflammation. To increase the frequency of pyelonephritis unilateral incomplete ureteral stenosis was created. The revertant strain assuming the hyphal form showed a significantly (p < 0.01) higher frequency of pyelonephritis as compared with the mutant strain not assuming this form, and the grade of inflammation was also higher in the revertant strain group. Also, higher renal tissue and serum levels of both lipid peroxide and superoxide dismutase, which are related to marked renal oxidant injury, tended to be correlated with the degree of neutrophil infiltration in the acute phase. These findings suggest that the hyphal form plays an important role in the development of C. albicans pyelonephritis and also that the oxygen radicals from neutrophils appearing at the sites of inflammation play a major part in the further extension of inflammatory lesions.

Molecular structure of the transposable element ninja in Drosophila simulans.

Genetically unstable DNA sequences of 16.1 kb in length were isolated from the white locus of the W(mky) strain of Drosophila simulans. This insertional DNA has some unique characteristics as a transposon. It is found in high numbers in this strain and its revertant strains W(psm1) and W(cho), but not elsewhere, and the sequence is a tandem triplication of a basic repeating unit. In order to determine the structure of the insert as whole and the functional unit as a transposon, we analyzed nine clones isolated from genomic libraries of W(psm1) and W(cho). The repeating unit of the 16.1 kb insertion was the retrotransposon ninja and the DNA sequence of the entire element was determined. The ninja transposon is 6644 bp in length, with a 316 bp long terminal repeat (LTR) on each end. It contains two openreading frames (ORFs), and the pol region is divided between the two ORFs in contrast the organization of other retrotransposons. An alignment analysis of the reverse transcriptase sequences suggested that the ninja element is the first Drosophila retrotransposon belonging to the Pao subgroup.

Reduced Virulence of Trichothecene-Nonproducing Mutants ofGibberella zeaein Wheat Field Tests

We have analyzed the role of trichothecene toxins in the virulence of the fungus Gibberella zeae (anamorph, Fusarium graminearum) on wheat (Triticum aestivum). Trichothecene-nonproducing mutants of G. zeae were obtained by disrupting Tri5, the gene encoding trichodiene synthase, which catalyzes the first committed step in the trichothecene biosynthetic pathway. Trichothecene-nonproducing mutants appear to be normal in growth and development under laboratory conditions. One such mutant was selfed to generate a meiotic revertant that lost the disruption vector and recovered trichothecene production. In the present study, virulence was assessed in 1994 and 1995 by controlled field inoculation of G. zeae spore suspensions into flowering wheat heads. Trichothecene-nonproducing (Tri5-) mutants were less virulent than the trichothecene-producing (Tri5+) parental and revertant strains in their ability to cause head scab on field-grown wheat. Although trichothecene-nonproducing strains colonized wheat heads, the infected heads showed less disease by several parameters we tested, including head bleaching symptoms, seed weight, seed viability, and trichothecene contamination. This evidence indicates that trichothecenes are virulence factors in wheat head scab.

Threonine dehydratases in different strains of Streptomyces fradiae

Among Streptomyces fradiae parent strain (NRRL 2702), aspartate auxotroph strain (SMF 305), and revenant strain (SMF 306), the revertant strain is the highest producer of tylosin and showed different repression patterns of tylosin production by ammonium ion from the parent strain. These results were elucidated by the facts that the revertant strain was superior to the parent or auxotroph strain in the biosynthesis of glutamine synthetase involved in ammonium assimilation and in the biosynthesis of threonine dehydratase (TDT) involved in providing precursors necessary for tylosin production, and ammonium ion inhibited the activity of TDT purified from the parent strain more than that of TDT from the revertant strain. TDT from the parent strain has been purified by DEAE cellulose, hydroxyapatite, Mono Q HR 5/5, and reversed-phase Protein C 4 chromatography. The molecular mass was 60 kDa by SDS-PAGE and 240 kDa by gel filtration. The N-terminal amino acid sequence of TDT was NH 3-E-A-T-G-P-L-T-T-E-S-G-A-P-V. The activity of TDT was allosterically activated by adenosine monophosphate.

The Essential Arginine Residue at Position 210 in the a Subunit of the Escherichia coli ATP Synthase Can Be Transferred to Position 252 with Partial Retention of Activity

The substitution of arginine at position 210 in the alpha subunit of Escherichia coli F0F1-ATPase by either lysine or alanine causes dominance in complementation tests with a chromosomal c subunit mutation. Reversal of dominance was achieved for the alpha R210K mutation but not for the alpha R210A mutation by the presence of an aspartic acid residue at position 50 or at position 252 in the alpha subunit. It was concluded that position 210 in putative helix 4 of a previously proposed model of the alpha subunit is close to position 252 in putative helix 5 and to position 50 in putative helix 1. The juxtaposition of residues 252 and 210 was also indicated by the observation that the double mutant alpha R210Q/Q252R was partially functional. A revertant of the partially functional double mutant, isolated on succinate medium, was found to contain a third mutation resulting in Pro-204 in the alpha subunit being replaced by threonine. That the revertant phenotype was due to the alpha P204T change was confirmed by site-directed mutagenesis. ATP synthesis in the revertant strain was at near normal levels as judged by growth yield experiments, but the revertant strain was unable to pump protons in response to ATP hydrolysis.

Thermosensitive UL9 gene function is required for early stages of herpes simplex virus type 1 DNA synthesis.

DNA replication of herpes simplex virus type 1 (HSV-1) is dependent on a virus-encoded sequence-specific origin-binding protein, the product of the UL9 reading frame. We have identified the mutations in the UL9 gene of three temperature-sensitive (ts) mutants of HSV-1 which are responsible for the ts phenotype (A90T in mutant tsS and V220M in tsR and tsX). The mutations are located in two different conserved helicase sequence motifs of UL9. Two further alterations (I204T and E280D) compared to the published sequence were found in the mutant, revertant and parental wild-type strain 17syn+ sequences and therefore seemed to be irrelevant for the ts phenotype. The ts function of the UL9 protein was required at early times during DNA synthesis whereas upward temperature shifts at later times did not considerably inhibit DNA synthesis.

Recovery of gene function by gene duplication in Saccharomyces cerevisiae.

A prototroph revertant (Rev9) selected from an ATCase- mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild-type gene. Sequence analysis of the 5' end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.

P element-induced mutations in Drosophila melanogaster showing high interspecific crossability between D. melanogaster females and D. simulans males.

Homozygous stocks for the second or the third chromosome of Drosophila melanogaster with a single insertional plwB element were screened for high crossability with D. simulans. Reciprocal crosses between each of these stocks and D. simulans were made, and the insemination rate at two or three days was examined. From two cycles of screening of the original 575 stocks, one stock (# 687) which showed high insemination rate was selected and was backcrossed to a w strain to substitute the background. We obtained a stock which showed 10% insemination rate with D. simulans males (control was 0%). No stocks exhibiting a high crossability with D. simulans females were acquired. Revertant strains, from which the P element had been lost, were obtained from the backcrossed # 687 stock. The insemination rates of 13 revertants to D. simulans males ranged from 1% to 33%. Seven of these 13 were not significantly different from the control line but were significantly different from the backcrossed # 687 stock. It was concluded that the mutation showing high crossability with D. simulans males was caused by the P element transposition.

Elements interrupting nitrogen fixation genes in cyanobacteria: presence and absence of a nifD element in clones of Nostoc sp. strain Mac

Nostocsp. strain Mac is capable of microaerobic, but not aerobic, nitrogen fixation (Fox). NostocMac grows as long, relatively straight, filaments that are well dispersed in the culture medium. However, spontaneouslγ-arising revertant strains selected for aerobic nitrogen fixation (Fox+) all grow as coiled filaments that associate in macroscopic clumps or balls of varying dimensions. DNA restriction fragment length polymorphism, using nitrogenase (nif) structural genes as probes, established identity between revertants and the parental culture. Mapping of the fragments and lack of hybridization to specific probes indicated the absence of a DNA sequence interrupting the nifDgene in one Fox+revertant. Such a nifDelement is assumed to be present in essentially all heterocyst-forming cyanobacteria. Only one clone out of 223 Fox-and Fox+ NostocMac clones surveyed lacked the nifDelement, indicating that loss of the element is a rare event. The nifDelement is present in the same location in the genome of NostocMac as it is in all other heterocyst-forming cyanobacteria analysed. No phenotypic differences could be detected between two Fox+clones containing or lacking the nifDelement, including repression and derepression of nitrogen fixation in response to the presence or absence of combined nitrogen. We suspect that retention of the nifDelement in vegetative cells of heterocyst-forming cyanobacteria is a consequence of selective pressure, although such selective conditions in laboratory cultures have not been identified.

Coordination of germ tube formation and surface antigen expression in Candida albicans

If the determinants of shape and cell wall topography are independently regulated and induced in germ tube formation in Candida albicans, these processes may be separable in a non-germ tube forming strain. The expression of several preferentially expressed hyphal surface components in a parental, non-germ tube forming variant, and a germ tube forming revertant strain were examined by indirect immunofluorescence. The proportion of germ tubes expressing the determinants and the morphological localization of expression was similar. Few yeast cells in germ tube cultures bound probes and there was no increase in binding by yeast cells of the variant strain. Extraction with β-mercaptoethanol prior to analysis had little effect on probe binding and the shape of yeast cells were similar. These observations suggest the ability to promote apical expansion in germ tube formation and surface expression of certain markers were coordinately regulated.

A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain

In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5'- and 3'-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe (r)) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe (r) phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (QA) to secondary quinone (QB) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (QA (-)).

Coordination of germ tube formation and surface antigen expression in Candida albicans.

If the determinants of shape and cell wall topography are independently regulated and induced in germ tube formation in Candida albicans, these processes may be separable in a non-germ tube forming strain. The expression of several preferentially expressed hyphal surface components in a parental, non-germ tube forming variant, and a germ tube forming revertant strain were examined by indirect immunofluorescence. The proportion of germ tubes expressing the determinants and the morphological localization of expression was similar. Few yeast cells in germ tube cultures bound probes and there was no increase in binding by yeast cells of the variant strain. Extraction with beta-mercaptoethanol prior to analysis had little effect on probe binding and the shape of yeast cells were similar. These observations suggest the ability to promote apical expansion in germ tube formation and surface expression of certain markers were coordinately regulated.

Intergenic suppression in a β subunit mutant with defective assembly in Escherichia coli F1ATPase

Substitution of Leu‐40 by Pro in the β subunit (βL40P) of Escherichia coli F1‐ATPase caused a decrease in the amount of the α and β subunits on the membranes. A revertant strain, Re50, carrying no suppression mutations in the uncD gene encoding the β subunit, was isolated from the βL40P mutant. The uncA gene from this revertant was amplified by PCR, and cloned into an expression plasmid. The expression plasmid carrying the uncA gene from the revertant was used for genetic suppression assays. The suppression mutation in Re50 was in the α subunit, and it recovered the assembly of the α and β subunits into the F1F0 complex and the ATPase activity to 50% that of the wild type. In Re50, Leu‐111 was substituted by Gln in the α subunit. These results suggest that the regions including Leu‐40 in the β subunit and Leu‐111 in the α subunit are located close together and interact with each other, either directly or indirectly.

Product-activation of Escherichia coli membrane-bound H +-ATPase (F 1F 0-ATPase) connected with ϵ-subunit at alkaline pH

Mutant strain AN1518 or AN2387 (Gly48 → Asp in ϵ-subunit) and partial revertant strain AN2540 (Gly48 → Asp, Pro47 → Ser in ϵ-subunit) of E. coli were used in a kinetic study of membrane-bound H +-ATPase. It was found that at pH 9.0 mutant strain AN1518 or AN2387 and partial revertant strain AN2540 gave a low initial rate, which increased with time until linearity was reached after 1–2 min. This phenomenon was prominent in mutant strains, but was not so obvious in wild-type AN346 of E. coli; this property is similar to F 1-ATPase reported by Cox [1]. The mechanism of the slow activation of membrane-bound H +-ATPase was further investigated in this paper. The experimental results indicated that the hydrolytic rate of E. coli F 1F 0-ATPase that increased with time was membrane protein concentration- and pH-dependent, and that the product ADP produced during ATP hydrolysis is the factor causing the slow activation. Preincubation of the hydrolytic product ADP with a concentration comparable to that produced in the assay (20 μM) caused initial activation of ATP hydrolysis and abolished the slow activation. On the other hand, with the removal of ADP during the progress of the hydrolytic reaction it could be seen that the slow activation was abolished as well. In order to test the relationship between the ϵ-subunit and ADP involved in the slow activation, trypsin treatment was carried out on the membrane-bound H +-ATPase of various strains. The activation observed after trypsin treatment was on the order of AN1518 > AN2540 > AN346. The activation effects of ADP and trypsin were not found to be additive. This implies that ADP acted in a similar way to trypsin, i.e., to cause removal of the ϵ-subunit. A tentative mechanism of the slow activation was proposed that ADP, a product of ATP hydrolysis, could induce conformational changes of F 1F 0 at alkaline pH 9.0, thus weakening the binding strength between the ϵ-subunit and other subunits of F 1F 0, and resulting in removal or partial removal of the ϵ-subunit. This further impaired the coupling of F 1 and F 0 in the mutant strains; as a consequence the rate of ATP hydrolysis was increased.

Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.

Further studies of clinical isolates of cysteine-requiring Escherichia coli and Klebsiella and possible mechanisms for their selection in vivo

Cysteine-dependent (cys-) Escherichia coli and Klebsiella spp., defective in sulphate assimilation, were isolated from urine and stool samples of infected patients. These isolates reverted to prototrophy under conditions of cysteine deprivation but the revertant strains and a prototrophic wild-type E. coli strain became auxotrophic for cysteine in a cysteine-enriched medium. This suggested that excess cysteine acts as a repressor of the cys HIJ operon known to control aspects of cysteine biosynthesis. A group of mostly elderly patients infected with cys- strains suffered a disproportionate amount of renal impairment as compared with a control group. In renal impairment, sulphur compounds, including cysteine, are retained. This raises the possibility that these raised levels of cysteine and related compounds may enhance the selection of cys- strains in vivo.

Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated proton transport.

Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness

The composition and positional distribution of fatty acids in the polar lipids from 4 strains of Chlorella differing in chilling susceptibility and frost hardiness were analyzed by enzymatic hydrolysis and gas‐liquid chromatography. Analysis of the polar lipids from chilling‐sensitive, chilling‐resistant and chilling‐sensitive revertant strains of Chlorella ellipsoidea IAM C‐102 showed that the sum of palmitic and trans‐3‐hexadecenoic acid in phosphatidylglycerol (PG) is about 60% for the sensitive strains and 53% for the resistant strain. The sum of dipalmitoyl and 1‐palmitoyl‐2‐(trans‐3‐hexadecenoyl) PG as estimated from the positional distribution of their fatty acids, is about 10% in the case of each of the three strains. The contents of unsaturated fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were higher in the resistant than in the sensitive strain. This suggests that unsaturation of fatty acids in not only PG but also PC and PE is involved in chilling sensitivity of Chlorella. On the other hand, lipid changes during the development of frost hardiness of C. ellipsoidea IAM C‐27, a frost hardy strain, were examined. The results showed that fatty acids in most lipid classes are unsaturated in the hardening process but their degree of unsaturation is not greatly different from that of the chilling‐resistant strain, suggesting that not only unsaturation of fatty acids in lipids but also other factors are necessary for the development of frost hardiness.

Reversion analysis of dynein intermediate chain function.

The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5' or 3') and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.

Sensitivity of Neurospora crassa to benzimidazoles and N-phenylcarbamates: Effect of amino acid substitutions at position 198 in β-tubulin

Diethofencarb-resistant revertants have been isolated from the Neurospora crassa mutant F914 which shows both carbendazim (MBC) resistance and diethofencarb sensitivity. Revertant strains FR422 and FR424 were true revertants showing high resistance to N-phenylcarbamates and sensitivity to benzimidazoles, the same as wild-type. However, other revertant strains, FR511, FR513, FR521, FR421, and FRB3, were more resistant to diethofencarb than the strain F914 but less resistant to diethofencarb than the wild-type strain. These moderately resistant strains were also more resistant to benzimidazoles than the parent strain F914, Strains FR421 and FR521 were also resistant to griseofulvin. All the diethofencarb-resistant mutations were linked to the Bml locus that encodes β-tubulin. The β-tubulin gene of FRB3 strain was cloned and sequenced, and an amino acid substitution at the position 198 was found. In the strains FRB3, F914, and wild-type, the amino acids at position 198 were Lys, Gly, and Glu, respectively. A new β-tubulin gene with Ala at position 198 was constructed by site-directed mutagenesis. This gene transformed the wild-type strain to MBC resistance, while transformation of the F914 strain with this gene resulted in growth on the medium containing 0.5 μg/ml diethofencarb but not on that containing 75 μg/ml diethofencarb. The F914 strain transformed by β-tubulin genes of the wild-type strain and the FRB3 strain grew even on medium containing 75 μg/ml diethofencarb. These data suggest that the presence of Glu at position 198 in β-tubulin is responsible for MBC sensitivity. Different amino acid substitutions at position 198 confer different sensitivity to diethofencarb and the order of amino acids which result in increasing sensitivity are Glu, Lys, Ala, and Gly (most sensitive). These approaches should work in clarifying the function of β-tubulin and also aid in the design of new potent fungicides.