One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

Untergasser, Andreas; Bijl, Gerben; Liu, Wei; Bisseling, Ton; Schaart, Jan G.; Geurts, René

doi:10.1371/journal.pone.0047885

Cited by 40 publications

(20 citation statements)

References 57 publications

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“…The uncovered evolutionary trajectory of a rhizobium nitrogen-fixing symbiosis provides novel leads in attempts to engineer nitrogen-fixing root nodules in agricultural crop plants. Such a translational approach is anticipated to be challenging 65 , and the only published study so far, describing transfer of 8 LCO signaling genes, was unsuccessful 66 . If we interpret the parallel loss of symbiosis genes in non-nodulating plants as evidence that these genes have been neofunctionalized to commit symbiotic functions, then this gene set is essential in any engineering approach.…”

Section: Discussionmentioning

confidence: 99%

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legumeParasponia

Velzen

Holmer

et al. 2017

Preprint

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30Rhizobium nitrogen-fixing nodules are a well-known trait of legumes, but nodules also occur 31 in other plant lineages either with rhizobium or the actinomycete Frankia as microsymbiont. 32The widely accepted hypothesis is that nodulation evolved independently multiple times, with 33 only a few losses. However, insight in the evolutionary trajectory of nodulation is lacking. We 34 conducted comparative studies using Parasponia (Cannabaceae), the only non-legume able This finding challenges a long-standing hypothesis on evolution of nitrogen-fixing symbioses, 42and has profound implications for translational approaches aimed at engineering nitrogen-43 fixing nodules in crop plants.

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Section: Discussionmentioning

confidence: 99%

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legumeParasponia

Velzen

Holmer

et al. 2017

Preprint

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“…To identify a line homozygous for a single transgene using these dominant markers it is necessary to: (1) plant a large number of T1 seeds and treat with a selective agent to identify transgenic plants; (2) harvest T2 seeds from resistant plants and screen these families for lines segregating 3:1 for the selectable trait; (3) harvest seeds from five to six plants in families with single insertions; and (4) plant these T3 seeds to identify families homozygous for the insertion. In contrast, fluorescent reporters can be scored in dry seed (Stuitje et al 2003;Lu and Kang 2008;Shimada et al 2010;Untergasser et al 2012) and are dosage sensitive (Shimada et al 2010;this report). Using these markers, the procedure for identifying lines homozygous for single transgenic insertions consists of: (1) visually selecting transgenic T1 seeds prior to planting; (2) screening the F2 seed from these T1 plants to identify lines segregating 3:1 for the fluorescent transgene, and (3) planting a few (three to five) of the brightest seeds from these families to obtain homozygous lines.…”

Section: Discussionmentioning

confidence: 99%

Traffic Lines: New Tools for Genetic Analysis inArabidopsis thaliana

Rossidivito

et al. 2015

Genetics

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Genetic analysis requires the ability to identify the genotypes of individuals in a segregating population. This task is straightforward if each genotype has a distinctive phenotype, but is difficult if these genotypes are phenotypically similar or identical. We show that Arabidopsis seeds homozygous or heterozygous for a mutation of interest can be identified in a segregating family by placing the mutation in trans to a chromosome carrying a pair of seed-expressed green and red fluorescent transgenes (a "traffic line") that flank the mutation. Nonfluorescent seeds in the self-pollinated progeny of such a heterozygous plant are usually homozygous for the mutation, whereas seeds with intermediate green and red fluorescence are typically heterozygous for the mutation. This makes it possible to identify seedlings homozygous for mutations that lack an obvious seedling phenotype, and also facilitates the analysis of lethal or sterile mutations, which must be propagated in heterozygous condition. Traffic lines can also be used to identify progeny that have undergone recombination within a defined region of the genome, facilitating genetic mapping and the production of near-isogenic lines. We produced 488 transgenic lines containing single genome-mapped insertions of NAP:dsRED and NAP:eGFP in Columbia (330 lines) and Landsberg erecta (158 lines) and generated sets of traffic lines that span most regions of the Arabidopsis genome. We demonstrated the utility of these lines for identifying seeds of a specific genotype and for generating near-isogenic lines using mutations of WUSCHEL and SHOOTMERISTEMLESS. This new resource significantly decreases the effort and cost of genotyping segregating families and increases the efficiency of experiments that rely on the ability to detect recombination in a defined chromosomal segment.

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“…Such genetic modifications will require transfer of multiple Transformation of legume plants 583 genes or pathways. Recently, Untergasser et al,2012) developed a vector able to transfer eight genes in one-step by Agrobacteriummediated transformation. Genes essential for M. truncatula to establish a symbiosis with rhizobia were transferred to four nonleguminous species; strawberry, poplar, tomato and tobacco and all the transgenes were shown to be expressed in the root tissue of these non-legumes.…”

Section: Discussionmentioning

confidence: 99%

Transformation of leguminous plants to study symbiotic interactions

Iantcheva

Mysore²,

Ratet³

2013

Int. J. Dev. Biol.

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Legume plants are important in agriculture because they represent an important source of protein for human and animal consumption. This high protein content results from their capacity to use atmospheric nitrogen for their nutrition as a consequence of their symbiotic interaction with rhizobia. Understanding this interaction at the molecular level is a prerequisite for its better use in agriculture and for the long term objective of its transfer to other crops. Agrobacterium-mediated transformation is a tool of choice for studying this interaction and for unraveling the function of the different genes discovered through classical genetic approaches. However, legume plants are often recalcitrant to regeneration and transformation. This paper describes the technology developments (regeneration, transformation, insertion mutagenesis) related to Agrobacterium transformations that were established in the legume plants, as well as different examples of the technology developments or gene discoveries resulting from these studies. KEY WORDS: legume plant, agrobacterium, rhizobium, transformation, symbiosis Legumes and their importanceAmongst the mineral nutrients required by plants, nitrogen is the most important but its availability is unfortunately low in most soils. The intensive use of industrial nitrogen fertilizers in modern agriculture in the last 50 years has allowed the development of very productive crops, but their intensive use also resulted in eutrophication of continental waters including some areas in Europe. There is thus a strong need to reduce application of chemical nitrogen fertilizers and improve alternative nitrogen inputs (Graham and Vance 2003;Ferguson et al., 2010) in agriculture. A key contribution of legumes to sustainable agriculture and nitrogen cycle is their ability to fix atmospheric nitrogen in most agricultural ecosystems. They form specialized organs, the root nodules, in association with specific soil bacteria called rhizobia (including the genera Azorhizobium, Allorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium). In these organs the bacteria differentiate into a form called the bacteroids and catalyze the reduction of atmospheric nitrogen gas (N 2 ) into ammonia using the nitrogenase enzyme complex. This process is commonly referred as "symbiotic nitrogen Abbreviations used in this paper: AM, arbuscular mycorrhiza; N2, nitrogen gas; NF, Nod factor; TDZ, thidiazuron; T-DNA, agrobacterium tranfered DNA.feed, and vegetable oil. They represent the third largest group of angiosperms and are the second largest group of food and feed crops grown worldwide. These plants are cultivated on 12-15% of available arable land and are responsible for more than 25% of the world's primary crop production. Around 250 million tons of grain legumes are produced annually world-wide. The ability of legumes to enter into symbiosis with nitrogen-fixing rhizobia provides them with a unique advantage compared to other plant species. For this reason legumes are called "green manu...

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One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

Cited by 40 publications

References 57 publications

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legumeParasponia

Parallel loss of symbiosis genes in relatives of nitrogen-fixing non-legumeParasponia

Traffic Lines: New Tools for Genetic Analysis inArabidopsis thaliana

Transformation of leguminous plants to study symbiotic interactions

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