Rice Acetolactate Synthase Gene Research Articles

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%–6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Conferred Resistance to an Acetolactate Synthase-inhibiting Herbicide in Transgenic Tall Fescue (Festuca arundinacea Schreb.)

Herbicide-resistant turfgrass can be an efficient tool that will allow easier turf maintenance. Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways leading to the branched-chain amino acids, and amino acid substitutions in ALS have been known to confer resistance to ALS-inhibiting herbicides. A two-point mutated rice ALS gene [OsALS (dm)] has been shown to confer strong resistance to bispyribac-sodium (BS), an ALS-inhibiting herbicide. In this study, we introduced into turf-type tall fescue (Festuca arundinacea Schreb.) the OsALS (dm) gene by using Agrobacterium-mediated transformation for conferring herbicide resistance. Stable integration of the transgene was confirmed by Southern blot analysis. Transgenic and wild-type plants were sprayed on the leaves with herbicide containing BS; approximately half of the transgenic plants were unaffected by the treatment and showed resistance to the herbicide, whereas the wild-type plants died. ALS activity in the leaf tissue of transgenic-resistant plants incubated with BS was almost equivalent to that in wild-type plants without BS and was higher than in wild-type plants incubated with BS. These indicate that the transgenic-resistant plants actively produced OsALS (dm) protein under herbicide treatment. This is the first report of herbicide-resistant transgenic tall fescue after introduction of a mutated ALS gene.

Development and application of allele-specific PCR assays for imazethapyr resistance in rice (Oryza sativa)

Outcrossing or cross hybridization is a potential concern in herbicide-resistant crop management strategies such as in the Clearfield™ rice system. Recent studies have shown that the mutated acetolactate synthase (ALS) gene that confers resistance to imazethapyr (Newpath) herbicide can be transferred from Clearfield rice cultivars via cross pollination under field conditions to weedy red rice. Resistance of commercial Clearfield rice cultivars to imazethapyr is due to the presence of two point mutations in the ALS gene that result in amino acid substitutions from serine to asparagine (S to D) and glycine to glutamic acid (G to E). We report here development of a DNA-based method that involves application of allele-specific PCR assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes produced from natural outcrosses between Clearfield varieties CL121, CL141, CL161 and weedy red rice. PCR assays that can distinguish between the homozygous and heterozygous imazethapyr-resistant S653D and G654E SNP alleles of the rice ALS gene were developed and evaluated. A total of 483 individual red rice plants were successfully screened for the presence of S653D SNP and another 145 F2 individuals from natural red rice × CL121 hybridizations were screened for the presence of the G654E SNP. The PCR-based assays produced during this study are simple, rapid, inexpensive, reproducible and require only standard PCR and electrophoretic instruments that can be applied toward outcrossing evaluation and effective weed management strategies for the Clearfield crop system.

Molecular breeding of a novel herbicide‐tolerant rice by gene targeting

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

Mutations in the red rice ALS gene associated with resistance to imazethapyr

The introduction of Clearfield (CL) rice cultivars resistant to imidazolinone herbicides, acetolactate synthase (ALS) inhibitors, has raised concerns of gene flow to weedy rice genotypes collectively called “red rice” that infest rice-growing areas in the southern United States. This experiment was conducted to study hybridization between CL rice and red rice using simple sequence repeats (SSR) markers, identify mutations in the ALS gene of imazethapyr-resistant red rice, and to detect the introgression of the ALS-resistant gene from CL rice into red rice. Natural outcrossing experiments between CL rice and strawhull (SH) red rice were set up in Stuttgart, AR, in 2002 and 2003. Putative red rice hybrids were detected among volunteer plants in the following year. Hybridization was confirmed using SSR markers, and introgression of the resistant ALS gene from CL rice to red rice was detected by ALS gene sequencing. The ALS gene sequences of U.S. rice cultivars ‘Bengal’ and ‘Cypress’, SH red rice, CL rice (CL161), and imazethapyr-resistant red rice/CL rice hybrids were compared. Nucleotide sequences of the ALS gene from the rice cultivars were identical. Three point mutations were present in the SH red rice ALS gene coding region relative to Bengal/Cypress. One of these resulted in the substitution of Asp630for Glu630. The ALS gene sequences of confirmed hybrids were identical to that of the herbicide-resistant pollen source, CL161. We identified four ALS gene mutations in the herbicide-resistant red rice hybrids relative to the susceptible rice cultivars. One point mutation, resulting in a substitution of Ser653with Asn, was linked to ALS resistance in callus tissue derived from a Kinmaze rice line from Japan. The other three mutations (Ser186—Pro, Lys416—Glu, and Leu662—Pro) are novel. This experiment confirmed that gene flow from imidazolinone-resistant rice resulted in herbicide-resistant red rice plants.