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.

Molecular basis of multiple resistance to herbicides inhibiting acetyl-CoA carboxylase and acetolactate synthase in American sloughgrass (Beckmannia syzigachne) from China

American sloughgrass (Beckmannia syzigachne Steud.) is a problematic grass that is widely distributed in wheat and oilseed rape fields in China. The herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl failed to control B. syzigachne JCWJ-R populations collected from a wheat field in Jiangsu Province. Dose-response experiments showed that JCWJ-R was resistant to the acetyl-CoA carboxylase (ACCase) inhibitors fenoxaprop-P-ethyl (33.8-fold), haloxyfop-R-methyl (12.7-fold), clethodim (7.8-fold) and pinoxaden (11.6-fold), and to the acetolactate synthase (ALS) inhibitors mesosulfuron-methyl (15.9-fold), pyroxsulam (17.6-fold), flucarbazone-Na (10.7-fold) and imazethapyr (7-fold). Resistance to ALS inhibitors was due to a Pro-197-Ser mutation in the ALS gene and resistance to ACCase inhibitors was due to an Ile-1781-Leu mutation in the ACCase gene. A derived cleaved amplified polymorphic sequence method was developed to detect the ALS mutation in B. syzigachne. This was combined with a previously established method to detect Ile-1781-Leu, and the mutation frequency and homozygous mutation rates in the JCWJ-R population were determined. The evolution of multiple resistance to ACCase and ALS inhibitors in this B. syzigachne population indicated that alternative methods should be developed to control resistant weeds.

Biallelic Gene Targeting in Rice.

Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained.

Acetolactate Synthase–Inhibiting, Herbicide-Resistant Rice Flatsedge (Cyperus iria): Cross-Resistance and Molecular Mechanism of Resistance

Overuse of acetolactate synthase (ALS)–inhibiting herbicides in rice has led to the evolution of halosulfuron-resistant rice flatsedge in Arkansas and Mississippi. Resistant accessions were cross-resistant to labeled field rates of ALS-inhibiting herbicides from four different families, in comparison to a susceptible (SUS) biotype. Resistance index of Arkansas and Mississippi accessions based on an R/S ratio of the lethal dose required for 50% plant mortality (LD50) to bispyribac-sodium, halosulfuron, imazamox, and penoxsulam was ≥ 21-fold. Control of Arkansas, Mississippi, and SUS accessions with labeled field rates of 2,4-D, bentazon, and propanil was ≥ 93%. An enzyme assay revealed that an R/S ratio for 50% inhibition (I50) of ALS for halosulfuron was 2,600 and 200 in Arkansas and Mississippi, respectively. Malathion studies did not reveal enhanced herbicide metabolism in resistant plants. The ALS enzyme assay and cross-resistance studies point toward altered a target site as the potential mechanism of resistance. Trp574–Leu amino acid substitution within the ALS gene was found in both Arkansas and Mississippi rice flatsedge accessions using the Illumina HiSeq platform, which corresponds to the mechanism of resistance found in many weed species. Field-rate applications of 2,4-D, bentazon, and propanil can be used to control these ALS-resistant rice flatsedge accessions.

Occurrence Cause, Mechanism and Control of Herbicide-Resistant Weeds in Rice Field of Korea

Sulfonylurea (SU)-resistant weeds of eight annual weeds, Monochoria vaginalis, Scirpus juncoides and Cyperus difformis, etc., and four perennial weeds, Scirpus planiculmis, Sagittaria pigmaea, Eleocharis acicularis and Sagittaria trifolia as of 2013 since identification Monochoria korsakowii in the reclaimed rice field in 1998. And the resistant Echinochloa oryzoides to ACCase and ALS inhibitors has been confirmed in rice fields of the southern province, Korea in 2009. In the beginning, the M. vaginalis, S. juncoides and C. difformis of these SU-resistant weeds were rapidly and individually spreaded in different fields, however, these resistant weeds have been occurring simultaneously in the same filed recently. The resistant biotype by weed species demonstrated about 10- to 1,000-fold resistance, based on GR50 values of the SU herbicides tested. And the resistant biotype of E. oryzoides to cyhalofop-butyl, pyriminobac-methyl, and penoxsulam was about 14, 8, and 11 times more resistant than the susceptible biotype base on GR50 values. The products and applied area of SU-included herbicides have been increased rapidly, and have accounted for about 69% and 96% in Korea, respectively. In Korea, the main cause of SU-resistant weed is extensive use of these herbicides. The top ten herbicides by applied area were composed of all SU-included herbicides by 2003. The concentrated and successive treatment of ACCase and ALS inhibitors for control of barnyardgrass in rice led up to the resistance of E. oryzoides. Also, SU-herbicides like pyrazosulfuron-ethyl and imazosulfuron which effective to barnyardgrass can be bound up with the resistance of E. oryzoides. The ALS activity isolated from the resistant biotype of M. korsakowii to SU-herbicides tested was less sensitive than that of susceptible biotype. The concentration of herbicide required for 50% inhibition of ALS activity (I50) of the SU-resistant M. korsakowii was 14- to 76-fold higher as compared to the susceptible biotype. No differences were observed in the rates of ( 14 C)bensulfuron uptake and translocation. Acetolactate synthase (ALS) genes from M. vaginalis resistant and susceptible biotypes against SU-herbicides revealed a single amino acid substitution of proline (CCT), at 197th position based on the M. korsakowii ALS sequence numbering, to serin (TCT) in conserved domain A of the gene. Carfentrazone-ethyl and pyrazolate were used mainly to control SU-resistant M. vaginalis by 2006 in Korea. However, the alternative herbicides such as benzobicyclone, to be possible to control simultaneously the several resistant weeds, have been developing and using broadly, because the several resistant weeds have been occurring simultaneously in the same fieled. The top ten herbicides by applied area in Korea have been occupied by products of 3-way mixture type including herbicides with alternative mode of action for the herbicide resistant weeds. Mefenacet, fentrazamide and cafenstrole had excellent controlling effects on the ACCase and ALS inhibitors resistant when they were applied within 2nd leaf stage.

English

The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters.

SNP markers for acetolactate synthase genes from tribenuron-methyl resistant mutants in Brassica napus L.

SNP markers for acetolactate synthase genes from tribenuron-methyl resistant mutants in Brassica napus L.

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

Main ConclusionThis is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides.Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2399-9) contains supplementary material, which is available to authorized users.

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.

Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.

Seed dormancy is modulated in recently evolved chlorsulfuron-resistant Turkish biotypes of wild mustard (Sinapis arvensis).

Biotypes of the broad-leaved wild mustard (Sinapis arvensis L.) found in wheat fields of Aegean and Marmara region of Turkey were characterized and shown to have developed resistance to sulfonylurea (chlorsulfuron), an inhibitor of acetolactate synthase (ALS). DNA sequence analysis of the ALS genes from two such resistant (“R”) biotypes, KNF-R1 and KNF-R2, revealed point mutations, CCT (Pro 197) to TCT (Ser 197) in KNF-R1 and CCT (Pro 197) to ACT (Thr 197) in KNF-R2; these substitutions are consistent with the presence of chlorsulfuron-insensitive ALS enzyme activity in the “R” S. arvensis biotypes. An additional phenotype of chlorsulfuron resistance in the Turkish S. arvensis “R” biotypes was revealed in the form of an altered seed dormancy behavior over 4–48 months of dry storage (after-ripening) compared to the susceptible (“S”) biotypes. Seeds of the “S” biotypes dry stored for 4 months had a higher initial germination, which sharply decreased with storage time, while the seeds of the “R” biotypes had lower germination after 4-months storage, rising sharply and peaking thereafter by 24 months' of dry storage. The “R” biotype seeds continued to maintain a higher germination percentage even after 48 months of after-ripening. The seed weight of “R” and “S” biotypes after-ripened for 4 months was similar but those after-ripened for 48 months differed, “R” seeds were significantly heavier than those of the “S” seeds. Differential seed germinability between “S” and “R” biotypes was found not a case of differential viability, temperature regimen or non-response to pro-germination hormone GA3. These studies are of relevance to ecological fitness of herbicide-resistant biotypes in terms of seed viability and germination.

Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose–response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

Cloning, expression, and characterization of an acetolactate synthase (ALS) gene from Anabaena azotica

A novel acetolactate synthase (ALS) gene was cloned from Anabaena azotica, which was isolated from China and is regarded as a vital and beneficial photosynthetic microorganism that can contribute to soil fertility and maintain ecosystem stability. The full-length gene consists of an open reading frame (ORF) of 1644bp and encodes a 59.2kDa 547 amino acid protein. Sequence alignment using the BLAST similarity search in the GenBank database revealed that the amino acid sequence was similar to ALS derived from Anabaena sp. PCC 7120. The ALS gene was subcloned into the plasmid pET28a (+) and expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant protein was purified and subsequently characterized. The properties of ALS from the E. coli transformant are similar to that of an enzyme from the original A. azotica. Conserved amino acid sequence and the predicted structure of the active site were consistent with those observed for the bacterial enzyme Klebsiella pneumoniae ALS in similar experiments. Our results represent a significant functional demonstration of the existence of an algae ALS subunit and provide a solid basis for further studies on the structure, function and properties of ALS, as well as its response mechanism to inhibitors.

Field-evolved resistance to four modes of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population.

Evolution of multiple herbicide resistance in weeds is a serious threat to weed management in crop production. Kochia is an economically important broadleaf weed in the U.S. Great Plains. This study aimed to confirm resistance to four sites of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population from a crop field near Garden City (GC), Kansas, and further determine the underlying mechanisms of resistance. One-fourth of the GC plants survived the labeled rate or higher of atrazine [photosystem II (PSII) inhibitor], and the surviving plants had the Ser-264 to Gly mutation in the psbA gene, the target site of atrazine. Results showed that 90% of GC plants survived the labeled rate of dicamba, a synthetic auxin. At least 87% of the plants survived up to 72 g a.i. ha(-1) of chlorsulfuron [acetolactate synthase (ALS) inhibitor], and analysis of the ALS gene revealed the presence of Pro-197 to Thr and/or Trp-574 to Lue mutation(s). Most GC plants also survived the labeled rate of glyphosate [5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor), and the resistant plants had 5-9 EPSPS gene copies (relative to the ALS gene). We confirm the first case of evolution of resistance to four herbicide sites of action (PSII, ALS and EPSPS inhibitors and synthetic auxins) in a single kochia population, and target-site-based mechanisms confer resistance to atrazine, glyphosate and chlorsulfuron.

Mutation at residue 376 of ALS confers tribenuron-methyl resistance in flixweed (Descurainia sophia) populations from Hebei Province, China

The acetolactate synthase (ALS) inhibitor tribenuron has been used continuously for approximately twenty years as an herbicide in winter wheat fields in China. Flixweed (Descurainia sophia) has evolved resistance to tribenuron, due to multiple amino acid mutations at the 197th residue of ALS. In this study, the molecular basis of tribenuron resistance was investigated using two resistant populations, Xingtai (XT) and Shijiazhuang (SJ), and two susceptible populations, Cangzhou (CZ) and Handan (HD). Whole-plant tests and ALS activity assays showed that the two resistant populations were highly resistant to tribenuron. Targeted amplification of ALS genes from the four populations showed that there were two ALS genes in each population, and both of them were expressed in flixweed; the full coding lengths of the two ALS genes were 1998bp and 2004bp. Mutations related to tribenuron resistance in flixweed were located in only the 1998bp paralog. An ALS activity assay showed that the resistant population SJ displayed slight cross-resistance to florasulam, with a resistance factor of 4.81, but the resistant population XT did not have cross-resistance to florasulam. The resistant population XT was found to carry the previously reported mutation Pro197Ser, but the resistant population SJ carried a different mutation, Asp376Glu, known from other weeds but novel in flixweed. Our results demonstrated that multiple versions of ALS genes exist in flixweed and that mutations at multiple sites may result in ALS-inhibitor resistance in this weed.

Target-site resistance to bensulfuron-methyl in Sagittaria trifolia L. populations

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in northeast of China and cannot be controlled effectively by bensulfuron-methyl in recent years. In this study, two suspected resistant S. trifolia populations (R1 and R2) were collected in Liaoning province of China. Whole-plant dose–response studies showed that R1 and R2 were highly resistant to bensulfuron-methyl, with the GR50 R/S ratios of 76.99 and 49.94 respectively. In vitro acetolactate synthase (ALS) assays revealed that resistance was due to reduced sensitivity of the ALS to bensulfuron-methyl inhibition, with I50 R/S ratios of 81.86 and 67.48 for R1 and R2, respectively. Total ALS activity was similar for the S and R2 populations, whereas the R1 population displayed significantly higher ALS activity than did the S population. The mutations Pro-197-Leu and Pro-197-Ser were identified in the ALS gene of the R1 and R2 populations, respectively. This is the first report examining bensulfuron-resistant S. trifolia in Liaoning province, China. The Pro197 mutation is likely responsible for resistance to bensulfuron-methyl in S. trifolia populations.

Multiple-herbicide resistance in Echinochloa crus-galli var. formosensis, an allohexaploid weed species, in dry-seeded rice

Biotypes of Echinochloa crus-galli var. formosensis with resistance to cyhalofop-butyl, an acetyl-CoA carboxylase (ACCase) inhibitor, have been found in dry-seeded rice fields in Okayama, Japan. We collected two lines with suspected resistance (Ecf27 and Ecf108) from dry-seeded rice fields and investigated their sensitivity to cyhalofop-butyl and other herbicides. Both lines exhibited approximately 7-fold higher resistance to cyhalofop-butyl than a susceptible line. Ecf108 was susceptible to penoxsulam, an acetolactate synthase (ALS) inhibitor. On the other hand, Ecf27 showed resistance to penoxsulam and two other ALS inhibitors: propyrisulfuron and pyriminobac-methyl. The alternative herbicides butachlor, thiobencarb, and bispyribac-sodium effectively controlled both lines. To examine the molecular mechanisms of resistance, we amplified and sequenced the target-site encoding genes in Ecf27, Ecf108, and susceptible lines. Partial sequences of six ACCase genes and full-length sequences of three ALS genes were examined. One of the ACCase gene sequences encodes a truncated aberrant protein due to a frameshift mutation in both lines. Comparisons of the genes among Ecf27, Ecf108, and the susceptible lines revealed that none of the ACCases and ALSs in Ecf27 and Ecf108 have amino acid substitutions that are known to confer herbicide resistance, although a single amino acid substitution was found in each of three ACCases in Ecf108. Our study reveals the existence of a multiple-herbicide resistant biotype of E. crus-galli var. formosensis at Okayama, Japan that shows resistance to cyhalofop-butyl and several ALS inhibitors. We also found a biotype that is resistant only to cyhalofop-butyl among the tested herbicides. The resistance mechanisms are likely to be non-target-site based, at least in the multiple-herbicide resistant biotype.

Responses to halosulfuron-methyl and flazasulfuron and mutation of acetolactate synthase gene of Cyperus brevifolius survived in turf grass on golf course

Responses to halosulfuron-methyl and flazasulfuron and mutation of acetolactate synthase gene of <i>Cyperus brevifolius</i> survived in turf grass on golf course

First report of resistance to acetolactate-synthase-inhibiting herbicides in yellow nutsedge (Cyperus esculentus): confirmation and characterization.

Yellow nutsedge is one of the most problematic sedges in Arkansas rice, requiring the frequent use of halosulfuron (sulfonylurea) for its control. In the summer of 2012, halosulfuron at 53 g ha(-1) (labeled field rate) failed to control yellow nutsedge. The level of resistance to halosulfuron was determined in the putative resistant biotype, and its cross-resistance to other acetolactate synthase (ALS) inhibitors from four different herbicide families. ALS enzyme assays and analysis of the ALS gene were used to ascertain the resistance mechanism. None of the resistant plants was killed by halosulfuron at a dose of 13 568 g ha(-1) (256× the field dose), indicating a high level of resistance. Based on the whole-plant bioassay, the resistant biotype was not controlled by any of the ALS-inhibiting herbicides (imazamox, imazethapyr, penoxsulam, bispyribac, pyrithiobac-sodium, bensulfuron and halosulfuron) tested at the labeled field rate. The ALS enzyme from the resistant biotype was 2540 times less responsive to halosulfuron than the susceptible biotype, and a Trp574 -to-Leu substitution was detected by ALS gene sequencing using the Illumina HiSeq. The results suggest a target-site alteration as the mechanism of resistance in yellow nutsedge, which accounts for the cross-resistance to other ALS-inhibiting herbicide families.

Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.