α-Tubulin Missense Mutations Correlate with Antimicrotubule Drug Resistance in Eleusine indica

Abstract

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three ␣ -tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) ␣ -tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an ␣ -tubulin gene confers resistance to dinitroanilines in goosegrass. INTRODUCTIONGoosegrass ( Eleusine indica ) is considered one of the most troublesome weeds in the world and occurs extensively in croplands of cotton and soybean and in turf in the southeastern United States (Mudge et al., 1984;Vaughn et al., 1990). This weed has been effectively controlled by preemergence application of dinitroaniline herbicides. However, repeated application in monocrop production has "selected" for a highly resistant (R) biotype from previously susceptible (S) biotype populations (Mudge et al., 1984). Although not as common, an intermediately resistant (I) biotype also has been reported (Mudge et al., 1984;Vaughn et al., 1990).Antimitotic dinitroanilines interfere with microtubule assembly, forming a tubulin-dinitroaniline complex that disrupts polymerization and microtubule stability (Morejohn et al., 1987;Hugdahl and Morejohn, 1993). In the unicellular alga Chlamydomonas reinhardtii , dinitroanilines shorten flagella and inhibit flagellar regeneration (Quader and Filner, 1980). In the parasitic protozoa Leishmania mexicana and Trypanosoma brucei , dinitroaniline apparently binds to protozoan tubulins and inhibits proliferation of these parasites (Chan and Fong, 1990;Chan et al., 1993). In higher plants, dinitroanilines work by affecting root growth, especially the development of secondary roots in susceptible plants (Hacskayko and Amato, 1968). In the presence of the herbicide, mitosis in the goosegrass S biotype is arrested at prometaphase, cell plates are misshapen or absent, and cells enlarge isodi...

Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

“…The weed goosegrass rapidly acquires resistance to dinitroaniline herbicides (Anthony et al, 1998Yamamoto et al, 1998;Zeng and Baird, 1999). These studies demonstrated that the ␣-tubulin mutations Thr239Ile and Met268Thr were associated with resistance to dinitroanilines and that the double point mutation (not observed in the original lines) produced higher oryzalin resistance than either of the single mutations alone.…”

“…The mutated amino acids in ␣-tubulin were identified within the modeled Toxoplasma ␣-tubulin structure. Two previously identified plant mutations Tyr24His and Met268Thr that confer dinitroaniline resistance were also included in our analysis (James et al, 1993;Yamamoto et al, 1998). The majority of the point mutations clustered within the core of ␣-tubulin ( Figure 4B).…”

“…These mutations function by hyperstabilizing microtubules and are not dinitroaniline specific (Schibler and Huang, 1991). The ␣-tubulin mutation Tyr24His was identified as a "step-up" mutation in a Chlamydomonas strain with low levels of dinitroaniline resistance and is the only specific dinitroaniline-resistance allele associated with tubulin (James et al, 1993).The weed goosegrass rapidly acquires resistance to dinitroaniline herbicides (Anthony et al, 1998Yamamoto et al, 1998;Zeng and Baird, 1999). These studies demonstrated that the ␣-tubulin mutations Thr239Ile and Met268Thr were associated with resistance to dinitroanilines and that the double point mutation (not observed in the original lines) produced higher oryzalin resistance than either of the single mutations alone.…”

Molecular Bases for Sensitivity to Tubulin-Binding Herbicides in Green Foxtail

“…Two distinct categories of resistant mutants, named R (resistant) and I (intermediate, moderately resistant), have been identified in this weed (for review, see . The molecular basis for resistance to dinitroanilines was found to be a Thr-239-Ile change in the a-tubulin TUA1 gene in the R mutant (Anthony et al, 1998;Yamamoto et al, 1998) and a Met-268-Thr change in the same gene in the I mutant (Yamamoto et al, 1998). In spite of comparison with the known threedimensional structure of the a,b-tubulin dimer from pig obtained by Nogales et al (1998), and although a recent modeling study proposed a binding site for tubulin-inhibiting herbicides in plants , the details of the interaction of tubulin-inhibiting herbicides with plant tubulins remain incompletely understood.…”

Cloning, Purification, and Polymerization ofCapsicum annuumRecombinant α and β Tubulin

“…These results were in good agreement with the aa sequences reported for plant tubulins, including Arabidopsis thaliana, 13,14) Glycine max L., 15) and Eleusine indica. 16,17) At the nucleotide level, the CAnm -and -TUB genes shared 78-95 and 76-93% homology with coding regions of plant tubulin cDNAs. 18) The genebank accession numbers for the sequences reported in C. annuum are EF495257 ( tubulin) and EF495259 ( tubulin).…”