scholarly journals Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

2021 ◽  
Author(s):  
Magdy Mahfouz ◽  
Haroon Butt ◽  
Jose Luis Moreno Ramirez
Keyword(s):  
Directed Evolution ◽  
Herbicide Resistance ◽  
Selection Pressure ◽  
High Efficiency ◽  
Rice Genome ◽  
Acetolactate Synthase ◽  
Target Sequence ◽  
T7 Promoter ◽  
Simultaneous Application ◽  
Dna Base

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated into the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as a selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

scholarly journals The Naturally Evolved EPSPS From Goosegrass Confers High Glyphosate Resistance to Rice

2021 ◽  
Vol 12 ◽  
Author(s):  
Chao Ouyang ◽  
Wei Liu ◽  
Silan Chen ◽  
Huimin Zhao ◽  
Xinyan Chen ◽  
...  
Keyword(s):  
Selection Pressure ◽  
High Efficiency ◽  
Rice Genome ◽  
Selection System ◽  
Transformation System ◽  
Wild Type ◽  
Promising Alternative ◽  
Two Generations ◽  
Massive Scale ◽  
Similar Growth

Glyphosate-resistant crops developed by the CP4-EPSPS gene from Agrobacterium have been planted on a massive scale globally, which benefits from the high efficiency and broad spectrum of glyphosate in weed control. Some glyphosate-resistant (GR) genes from microbes have been reported, which might raise biosafety concerns. Most of them were obtained through a hygromycin-HPT transformation system. Here we reported the plant source with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from goosegrass endowed rice with high resistance to glyphosate. The integrations and inheritability of the transgenes in the rice genome were investigated within two generations. The EiEPSPS transgenic plants displayed similar growth and development to wild type under no glyphosate selection pressure but better reproductive performance under lower glyphosate selection pressure. Furthermore, we reconstructed a binary vector pCEiEPSPS and established the whole stage glyphosate selection using the vector. The Glyphosate-pCEiEPSPS selection system showed a significantly higher transformation efficiency compared with the hygromycin-HPT transformation system. Our results provided a promising alternative gene resource to the development of GR plants and also extended the plant transformation toolbox.

Smallseed Falseflax (Camelina microcarpa) Management in Oklahoma Winter Wheat

2021 ◽  
pp. 1-14
Author(s):  
Jodie A. Crose ◽  
Misha R. Manuchehri ◽  
Todd A. Baughman
Keyword(s):  
Winter Wheat ◽  
Weed Control ◽  
Herbicide Resistance ◽  
Wheat Yield ◽  
Acetolactate Synthase ◽  
Growing Season ◽  
Time Of Application ◽  
Adequate Control ◽  
Growing Seasons

Abstract Three herbicide premixes have recently been introduced for weed control in wheat. These include: halauxifen + florasulam, thifensulfuron + fluroxypyr, and bromoxynil + bicyclopyrone. The objective of this study was to evaluate these herbicides along with older products for their control of smallseed falseflax in winter wheat in Oklahoma. Studies took place during the 2017, 2018, and 2020 winter wheat growing seasons. Weed control was visually estimated every two weeks throughout the growing season and wheat yield was collected in all three years. Smallseed falseflax size was approximately six cm in diameter at time of application in all years. Control ranged from 96 to 99% following all treatments with the exception of bicyclopyrone + bromoxynil and dicamba alone, which controlled falseflax 90%. All treatments containing an acetolactate synthase (ALS)-inhibiting herbicide achieved adequate control; therefore, resistance is not suspected in this population. Halauxifen + florasulam and thifensulfuron + fluroxypyr effectively controlled smallseed falseflax similarly to other standards recommended for broadleaf weed control in wheat in Oklahoma. Rotational use of these products allows producers flexibility in controlling smallseed falseflax and reduces the potential for development of herbicide resistance in this species.

Investigating the resistance levels and mechanisms to penoxsulam and cyhalofop-butyl in barnyardgrass (Echinochloa crus-galli) from Ningxia province, China

Weed Science ◽  
2021 ◽  
pp. 1-25
Author(s):  
Qian Yang ◽  
Xia Yang ◽  
Zichang Zhang ◽  
Jieping Wang ◽  
Weiguo Fu ◽  
...  
Keyword(s):  
Herbicide Resistance ◽  
Molecular Mechanisms ◽  
Acetolactate Synthase ◽  
Rice Fields ◽  
Target Site ◽  
Herbicide Resistant ◽  
Susceptible Populations ◽  
High Level ◽  
Als Inhibitor ◽  
Encoding Genes

Abstract Barnyardgrass (Echinochloa crus-galli) is a noxious grass weed which infests rice fields and causes huge crop yield losses. In this study, we collected twelve E. crus-galli populations from rice fields of Ningxia province in China and investigated the resistance levels to acetolactate synthase (ALS) inhibitor penoxsulam and acetyl-CoA carboxylase (ACCase) inhibitor cyhalofop-butyl. The results showed that eight populations exhibited resistance to penoxsulam and four populations evolved resistance to cyhalofop-butyl. Moreover, all of the four cyhalofop-butyl-resistant populations (NX3, NX4, NX6 and NX7) displayed multiple-herbicide-resistance (MHR) to both penoxsulam and cyhalofop-butyl. The alternative herbicides bispyribac-sodium, metamifop and fenoxaprop-P-ethyl cannot effectively control the MHR plants. To characterize the molecular mechanisms of resistance, we amplified and sequenced the target-site encoding genes in resistant and susceptible populations. Partial sequences of three ALS genes and six ACCase genes were examined. A Trp-574-Leu mutation was detected in EcALS1 and EcALS3 in two high-level (65.84- and 59.30-fold) penoxsulam-resistant populations NX2 and NX10, respectively. In addition, one copy (EcACC4) of ACCase genes encodes a truncated aberrant protein due to a frameshift mutation in E. crus-galli populations. None of amino acid substitutions that are known to confer herbicide resistance were detected in ALS and ACCase genes of MHR populations. Our study reveals the widespread of multiple-herbicide resistant E. crus-galli populations at Ningxia province of China that exhibit resistance to several ALS and ACCase inhibitors. Non-target-site based mechanisms are likely to be involved in E. crus-galli resistance to the herbicides, at least in four MHR populations.

scholarly journals Evaluation of Herbicide-Resistance Status on Populations of Littleseed Canarygrass (Phalaris Minor Retz.) from Southern Greece and Suggestions for their Effective Control

2012 ◽  
Vol 52 (3) ◽  
pp. 308-313 ◽  
Author(s):  
Ilias Travlos
Keyword(s):  
Herbicide Resistance ◽  
Resistance Index ◽  
Acetolactate Synthase ◽  
Effective Control ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Resistance Level ◽  
Phalaris Minor ◽  
Wide Range ◽  
Southern Greece

Evaluation of Herbicide-Resistance Status on Populations of Littleseed Canarygrass (Phalaris MinorRetz.) from Southern Greece and Suggestions for their Effective ControlIn 2010, a survey was conducted in the wheat fields of a typical cereal-producing region of Greece to establish the frequency and distribution of herbicide-resistant littleseed canarygrass (Phalaris minorRetz.). In total, 73 canarygrass accessions were collected and screened in a field experiment with several herbicides commonly used to control this weed. Most of the weed populations were classed as resistant (or developing resistance) to the acetyl-CoA varboxylase (ACCase)-inhibiting herbicide diclofop, while resistance to clodinafop was markedly lower. The results of the pot experiments showed that some of the canary populations were found to have a very high level of diclofop resistance (resistance index up to 12.4), while cross resistance with other herbicides was also common. The levels of resistance and cross resistance patterns among populations varied along with the different amounts and times of selection pressure. Such variation indicated either more than one mechanism of resistance or different resistance mutations in these weed populations. The population which had the highest diclofop resistance level, showed resistance to all aryloxyphenoxypropinate (APP) herbicides applied and non-ACCase inhibitors. Alternative ACCase-inhibiting herbicides, such as pinoxaden remain effective on the majority of the tested canarygrass populations, while the acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron + iodosulfuron could also provide some solutions. Consequently, there is an opportunity to effectively control canarygrass by selecting from a wide range of herbicides. It is the integration of agronomic practices with herbicide application, which helps in effective management ofP. minorand particularly its resistant populations.

scholarly journals Identification of Selection Signatures in Commercial Asian Rice Subspecies Deciphered Selection Footprints for Four Overrepresented Biological Processes

2020 ◽  
Author(s):  
Siavash Salek Ardestani ◽  
Mahmoud Amiri Roudbar ◽  
Mohammad Hossein Banabazi ◽  
Seyedeh Fatemeh Mousavi ◽  
Madhav Bhatta ◽  
...  
Keyword(s):  
Selection Pressure ◽  
Rice Genome ◽  
Fixation Index ◽  
Biological Processes ◽  
Selection Signatures ◽  
Genomic Changes ◽  
Asian Rice ◽  
Genome Level ◽  
Window Approach ◽  
Genomic Regions

Abstract BackgroundSelective breeding pressures have led to gradual genomic changes in Asian commercial rice, which have shaped selection footprints on its genome level. Tracing genomic selection footprints might be illuminative for better understanding of recent selection breeding objectives, and how breeding strategies have formed the Asian commercial rice genome. ResultsIn this study, the genotypic information (HDRA 700K) of four Asian commercial rice subspecies including Indica (n=498), Aus (n=187), Temperate japonica (n=241), and Tropical japonica (n=361) were downloaded from Rice Diversity Project database (http://www.ricediversity.org) to detect selection signatures by employing the Z-transformed of fixation index and Tajima’s D test, based on a sliding window approach. Although we could not identify overrepresented genomic regions underlying selection pressure among all aforementioned Asian commercial rice subspecies, interestingly, our findings revealed four overrepresented biological processes underlying selection pressure including proteolysis (GO:0006508), phosphorylation (GO:0016310), protein catabolic process (GO:0030163), and transmembrane transport (GO:0055085) that might be associated with immunity, senescing leaves, transporting, and absorption of ions. ConclusionsThese results can provide knowledge on how breeding efforts shaped the Asian commercial rice subspecies genome, and which genomic regions of these subspecies have been targeted in recent decades.

Herbicide resistance in China: a quantitative review

Weed Science ◽  
2019 ◽  
Vol 67 (6) ◽  
pp. 605-612 ◽  
Author(s):  
Xiangying Liu ◽  
Shihai Xiang ◽  
Tao Zong ◽  
Guolan Ma ◽  
Lamei Wu ◽  
...  
Keyword(s):  
Herbicide Resistance ◽  
Crop Production ◽  
Rapid Evolution ◽  
Acetolactate Synthase ◽  
Cross Resistance ◽  
Weed Species ◽  
Herbicide Resistant ◽  
Auxin Herbicides ◽  
Economic Boom ◽  
Meta Analyses

AbstractThe widespread, rapid evolution of herbicide-resistant weeds is a serious and escalating agronomic problem worldwide. During China’s economic boom, the country became one of the most important herbicide producers and consumers in the world, and herbicide resistance has dramatically increased in the past decade and has become a serious threat to agriculture. Here, following an evidence-based PRISMA (preferred reporting items for systematic reviews and meta-analyses) approach, we carried out a systematic review to quantitatively assess herbicide resistance in China. Multiple weed species, including 26, 18, 11, 9, 5, 5, 4, and 3 species in rice (Oryza sativa L.), wheat (Triticum aestivum L.), soybean [Glycine max (L.) Merr.], corn (Zea mays L.), canola (Brassica napus L.), cotton (Gossypium hirsutum L.)., orchards, and peanut (Arachis hypogaea L.) fields, respectively, have developed herbicide resistance. Acetolactate synthase inhibitors, acetyl-CoA carboxylase inhibitors, and synthetic auxin herbicides are the most resistance-prone herbicides and are the most frequently used mechanisms of action, followed by 5-enolpyruvylshikimate-3-phosphate synthase inhibitors and protoporphyrinogen oxidase inhibitors. The lack of alternative herbicides to manage weeds that exhibit cross-resistance or multiple resistance (or both) is an emerging issue and poses one of the greatest threats challenging the crop production and food safety both in China and globally.

Empirical investigation of mutation rate for herbicide resistance

Weed Science ◽  
2019 ◽  
Vol 67 (4) ◽  
pp. 361-368 ◽  
Author(s):  
Federico A. Casale ◽  
Darci A. Giacomini ◽  
Patrick J. Tranel
Keyword(s):  
Mutation Rate ◽  
Herbicide Resistance ◽  
De Novo ◽  
Selection Process ◽  
Theoretical Calculations ◽  
Acetolactate Synthase ◽  
De Novo Mutation ◽  
De Novo Mutations ◽  
Sources Of Resistance ◽  
Resistant Mutants

AbstractIn a predictable natural selection process, herbicides select for adaptive alleles that allow weed populations to survive. These resistance alleles may be available immediately from the standing genetic variation within the population or may arise from immigration via pollen or seeds from other populations. Moreover, because all populations are constantly generating new mutant genotypes by de novo mutations, resistant mutants may arise spontaneously in any herbicide-sensitive weed population. Recognizing that the relative contribution of each of these three sources of resistance alleles influences what strategies should be applied to counteract herbicide-resistance evolution, we aimed to add experimental information to the resistance evolutionary framework. Specifically, the objectives of this experiment were to determine the de novo mutation rate conferring herbicide resistance in a natural plant population and to test the hypothesis that the mutation rate increases when plants are stressed by sublethal herbicide exposure. We used grain amaranth (Amaranthus hypochondriacus L.) and resistance to acetolactate synthase (ALS)-inhibiting herbicides as a model system to discover spontaneous herbicide-resistant mutants. After screening 70.8 million plants, however, we detected no spontaneous resistant genotypes, indicating the probability of finding a spontaneous ALS-resistant mutant in a given sensitive population is lower than 1.4 × 10−8. This empirically determined upper limit is lower than expected from theoretical calculations based on previous studies. We found no evidence that herbicide stress increased the mutation rate, but were not able to robustly test this hypothesis. The results found in this study indicate that de novo mutations conferring herbicide resistance might occur at lower frequencies than previously expected.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

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