Srour A.,Southern Illinois University Carbondale |
Afzal A.J.,Southern Illinois University Carbondale |
Afzal A.J.,Ohio State University |
Blahut-Beatty L.,Agriculture and Agri Food Canada |
And 9 more authors.
BMC Genomics | Year: 2012
Background: Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O'Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS.Results: A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30-50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory.Conclusions: The inference that soybean has adapted part of an existing pathogen recognition and defense cascade (H.glycines; SCN and insect herbivory) to a new pathogen (F. virguliforme; SDS) has broad implications for crop improvement. Stable resistance to many pathogens might be achieved by manipulation the genes encoding a small number of pathogen recognition proteins. © 2012 Srour et al.; licensee BioMed Central Ltd.
« SLAC, U Toronto team develops new highly efficient ternary OER catalyst for water-splitting using earth-abundant metals; >3x TOF prior record-holder | Main | Six automated truck platoons to compete in European Truck Platooning Challenge » Researchers from the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) have designed and constructed of the first minimal synthetic bacterial cell, JCVI-syn3.0. Using the first synthetic cell, Mycoplasma mycoides JCVI-syn1.0 (created by this same team in 2010, earlier post), JCVI-syn3.0 was developed through a design, build, and test process using genes from JCVI-syn1.0. The new minimal synthetic cell contains 531,560 base pairs and just 473 genes, making it the smallest genome of any organism that can be grown in laboratory media. Of these genes 149 are of unknown biological function. By comparison the first synthetic cell, M. mycoides JCVI-syn1.0 has 1.08 million base pairs and 901 genes. A paper describing this research is being published in the journal Science by lead authors Clyde A. Hutchison, III, Ph.D. and Ray-Yuan Chuang, Ph.D., senior author J. Craig Venter, Ph.D., and senior team of Hamilton O. Smith, MD, Daniel G. Gibson, Ph.D., and John I. Glass, Ph.D. Our attempt to design and create a new species, while ultimately successful, revealed that 32% of the genes essential for life in this cell are of unknown function, and showed that many are highly conserved in numerous species. All the bioinformatics studies over the past 20 years have underestimated the number of essential genes by focusing only on the known world. This is an important observation that we are carrying forward into the study of the human genome. The research to construct the first minimal synthetic cell at JCVI was the culmination of 20 years of research that began in 1995 after the genome sequencing of the first free-living organism, Haemophilus influenza, followed by the sequencing of Mycoplasma genitalium. A comparison of these two genomes revealed a common set of 256 genes which the team thought could be a minimal set of genes needed for viability. In 1999 Dr. Hutchison led a team who published a paper describing the use of global transposon mutagenesis techniques to identify the nonessential genes in M. genitalium. Over the last 50 years more than 2,000 publications have contemplated minimal cells and their use in elucidating first principals of biology. From the start, the goal of the JCVI team was similar—build a minimal operating system of a cell to understand biology but to also have a desirable chassis for use in industrial applications. The creation of the first synthetic cell in 2010 did not inform new genome design principles since the M. mycoides genome was mostly recapitulated as in nature. Rather, it established a work flow for building and testing whole genome designs, including a minimal cell, from the bottom up starting from a genome sequence. To create JCVI-syn3.0, the team used an approach of whole genome design and chemical synthesis followed by genome transplantation to test if the cell was viable. Their first attempt to minimize the genome began with a simple approach using information in the biochemical literature and some limited transposon mutagenesis work, but this did not result in a viable genome. After improving transposon methods, they discovered a set of quasi-essential genes that are necessary for robust growth which explained the failure of their first attempt. To facilitate debugging of non-functional reduced genome segments, the team built the genome in eight segments at a time so that each could be tested separately before combining them to generate a minimal genome. The team also explored gene order and how that affects cell growth and viability, noting that gene content was more critical to cell viability than gene order. They went through three cycles of designing, building, and testing ensuring that the quasi-essential genes remained, which in the end resulted in a viable, self-replicating minimal synthetic cell that contained just 473 genes, 35 of which are RNA-coding. In addition, the cell contains a unique 16S gene sequence. The team was able to assign biological function to the majority of the genes with 41% of them responsible for genome expression information, 18% related to cell membrane structure and function, 17% related to cytosolic metabolism, and 7% preservation of genome information. However, a surprising 149 genes could not be assigned a specific biological function despite intensive study. This remains an area of continued work for the researchers. The team concludes that a major outcome of this minimal cell program are new tools and semi-automated processes for whole genome synthesis. Many of these synthetic biology tools and services are commercially available through SGI and SGI-DNA including a synthetic DNA construction service specializing in building large and complex DNA fragments including combinatorial gene libraries, Archetype genomics software, Gibson Assembly kits, and the BioXp, which is a benchtop instrument for producing accurate synthetic DNA fragments. Other authors on the paper are: Thomas J. Deerinck and Mark H. Ellisman, Ph.D., University of California, San Diego National Center for Microscopy and Imaging Research; James F. Pelletier, Center for Bits and Atoms and Department of Physics, Massachusetts Institute of Technology; Elizabeth A. Strychalski, National Institute of Standards and Technology. This work was funded by SGI, the JCVI endowment and the Defense Advanced Research Projects Agency’s Living Foundries program, HR0011-12-C-0063.
Electron micrograph image shows synthetic cell clusters of JCVI-syn3.0 cells magnified at about 15,000 times in this image released on March 24, 2016. REUTERS/Tom Deerinck and Mark Ellisman/The National Center for Imaging and Microscopy Research/University of California at San Diego/Handout via Reuters More WASHINGTON (Reuters) - Scientists on Thursday announced the creation of a synthetic organism stripped down to the bare essentials with the fewest genes needed to survive and multiply, a feat at the microscopic level that may provide big insights on the very nature of life. Genome research pioneer J. Craig Venter called the bacterial cell his research team designed and constructed the "most simple of all organisms." While the human genome possesses more than 20,000 genes, the new organism gets by with only 473. "This study is definitely trying to understand a minimal basis of life," said Venter. But the researchers said that even with such a simple organism, that understanding remained elusive. They noted that even though their organism has so few genes, they were still uncertain about the function of nearly a third of them, even after more than five years of work. The researchers predicted their work would yield practical applications in developing new medicines, biochemicals, biofuels and in agriculture. "Our long-term vision has been to design and build synthetic organisms on demand where you can add in specific functions and predict what the outcome is going to be," said Daniel Gibson, vice president for DNA technologies at Synthetic Genomics Inc, the company handling commercial applications from the research. "I think it's the start of a new era," Venter added. Venter helped map the human genome in 2001 and created the first synthetic cell in 2010 with the same team that conducted the new research. That 2010 achievement, creating a bacterial organism with a manmade genome, demonstrated that genomes can be designed on a computer, made in a laboratory and transplanted into a cell to form a new, self-replicating organism. Having created that synthetic cell, the researchers set out to engineer a bacterium by removing unessential genes. The goal was to use the fewest genes necessary for the organism to live and reproduce. Venter said initially "every one of our designs failed" because they took out too many genes, and had to restore some. Venter said one lesson was that to understand life, it is more important to look across the entirely of a genome, an organism's complete genetic blueprint, rather than at individual genes. "Life is much more like a symphony orchestra than a piccolo player. And we're applying the same philosophy now to our analysis of the human genome, where we're finding most human conditions are affected by variations across the entire genome" rather than a single gene, Venter said. The researchers said they created a minimal cell possessing the smallest genome of any self-replicating organism. They said a cell with even fewer genes could be possible although it might, for example, reproduce excruciatingly slowly. Microbiologist Clyde Hutchison of the J. Craig Venter Institute in La Jolla California, lead author of the study in the journal Science, said the goal is to figure out the functions of all the cell's genes and make a computer model to predict how it would grow and change in different environments or with additional genes. "It's important to realize there is no cell that exists where we know the functions of all the genes," Hutchison said. The environmental group Friends of the Earth expressed concern about the research, citing the lack of government regulations specific to synthetic biology and gene editing technologies. "Living organisms like bacteria are not machines to be rewired," said Dana Perls, an official of the group. "Not even the scientists know the biological function of 149 of these genes, which raises safety concerns. If we don't fully understand the science, it is more difficult to manage biosafety concerns." The research might shed light on the origins of life on Earth billions of years ago. "We may be getting hints of some early fundamental mechanisms that coincide with some of the most primitive kind of life forms," Venter said. "I think as we get the ability to explore further in the universe, my view is wherever we have the same chemical constituents, which is almost everywhere, life will happen," Venter said. "But that's a philosophical point until it's proven."
Genomics entrepreneur Craig Venter has created a synthetic cell that contains the smallest genome of any known, independent organism. Functioning with 473 genes, the cell is a milestone in his team’s 20-year quest to reduce life to its bare essentials and, by extension, to design life from scratch. Venter, who has co-founded a company that seeks to harness synthetic cells for making industrial products, says that the feat heralds the creation of customized cells to make drugs, fuels and other products. But an explosion in powerful ‘gene-editing’ techniques, which enable relatively easy and selective tinkering with genomes, raises a niggling question: why go to the trouble of making new life forms when you can simply tweak what already exists? Unlike the first synthetic cells made in 20101, in which Venter’s team at the J. Craig Venter Institute in La Jolla, California, copied an existing bacterial genome and transplanted it into another cell, the genome of the minimal cells is like nothing in nature. Venter says that the cell, which is described in a paper released on 24 March in Science2, constitutes a brand new, artificial species. “The idea of building whole genomes is one of the dreams and promises of synthetic biology,” says Paul Freemont, a synthetic biologist at Imperial College London, who is not involved in the work. The design and synthesis of genomes from scratch remains a niche pursuit, and is technically demanding. By contrast, the use of genome editing is soaring — and its most famous tool, CRISPR–Cas9, has already gained traction in industry, agriculture and medicine, notes George Church, a genome scientist at Harvard Medical School in Boston, Massachusetts, who works with CRISPR. “With much less effort, CRISPR came around and suddenly there are 30,000 people practising CRISPR, if not more.” Microbiologists were just starting to characterize the bacterial immune system that scientists would eventually co-opt and name CRISPR when Venter’s team began its effort to whittle life down to its bare essentials. In a 1995 Science paper, Venter’s team sequenced the genome of Mycoplasma genitalium, a sexually transmitted microbe with the smallest genome of any known free-living organism3, and mapped its 470 genes. By inactivating genes one by one and testing to see whether the bacterium could still function, the group slimmed this list down to 375 genes that seemed essential. One way to test this hypothesis is to make an organism that contains just those genes. So Venter, together with his close colleagues Clyde Hutchison and Hamilton Smith and their team, set out to build a minimal genome from scratch, by joining together chemically synthesized DNA segments. The effort required the development of new technologies, but by 2008, they had used this method to make what was essentially an exact copy of the M. genitalium genome that also included dozens of non-functional snippets of DNA ‘watermarks’4. But the sluggish growth of natural M. genitalium cells prompted them to switch to the more prolific Mycoplasma mycoides. This time, they not only synthesized its genome and watermarked it with their names and with famous quotes, but also implanted it into another bacterium that had been emptied of its own genome. The resulting ‘JCVI-syn1.0’ cells were unveiled1 in 2010 and hailed — hyperbolically, many say — as the dawn of synthetic life. (The feat prompted US President Barack Obama to launch a bioethics review, and the Vatican to question Venter’s claim that he had created life.) However, the organism’s genome was built by copying an existing plan and not through design — and its bloated genome of more than 1 million DNA bases was anything but minimal. In an attempt to complete its long-standing goal of designing a minimal genome, Venter’s team designed and synthesized a 483,000-base, 471-gene M. mycoides chromosome from which it had removed genes responsible for the production of nutrients that could be provided externally, and other genetic ‘flotsam’. But this did not produce a viable organism. So, in a further move, the team developed a ‘design-build-and-test’ cycle. It broke the M. mycoides genome into eight DNA segments and mixed and matched these to see which combinations produced viable cells; lessons learned from each cycle informed which genes were included in the next design. This process highlighted DNA sequences that do not encode proteins but that are still needed because they direct the expression of essential genes, as well as pairs of genes that perform the same essential task — when such genes are deleted one at a time, both mistakenly seem to be dispensable. Eventually, the team hit on the 531,000-base, 473-gene design that became known as JCVI-syn3.0 (syn2.0 was a less streamlined intermediary). Syn3.0 has a respectable doubling time of 3 hours, compared with, for instance, 1 hour for M. mycoides and 18 hours for M. genitalium. “This old Richard Feynman quote, ‘what I cannot create, I do not understand’, this principle is now served,” says Martin Fussenegger, a synthetic biologist at the Swiss Federal Institute of Technology (ETH) in Zurich, Switzerland. “You can add in genes and see what happens.” With nearly all of its nutrients supplied through growth media, syn3.0’s essential genes tend to be those involved in cellular chores such as making proteins, copying DNA and building cellular membranes. Astoundingly, Venter says that his team could not identify the function of 149 of the genes in syn3.0’s genome, many of which are found in other life forms, including humans. “We don’t know about a third of essential life, and we’re trying to sort that out now,” he says. This has blown Fussenegger away. “We’ve sequenced everything on this planet, and we still don’t know 149 genes that are most essential for life!” he says. “This is the coolest thing I want to know.” Syn3.0’s lasting impact on synthetic biology is an open question. “I think it’s kind of a George Mallory moment,” says Church, referring to the English mountaineer who died in 1924 trying to become the first person to climb Mount Everest. “‘Because it’s there’ was the excuse he gave for climbing Everest.” Church says that genome-editing techniques will remain the go-to choice for most applications that require a small number of genetic alterations, whereas genome design will be useful for specialized applications, such as recoding an entire genome to incorporate new amino acids. Fussenegger thinks that genome editing will be the favoured approach for therapies, but that writing genomes from scratch will appeal to scientists interested in fundamental questions about how genomes evolve, for instance. Even Venter acknowledges that syn3.0’s genome, although new, was designed by trial and error, rather than being based on a fundamental understanding of how to build a functioning genome. But he expects fast improvements, and thinks that genome synthesis from scratch will become the preferred approach for manipulating life. “If you want to make a few changes, CRISPRs are a great tool,” he says. “But if you’re really making something new and you’re trying to design life, CRISPRs aren’t going to get you there.”
Scientists have deleted nearly half the genes of a microbe, creating a stripped-down version that still functions, an achievement that might reveal secrets of how life works. It may also help researchers create new bacteria tailored for pumping out medicines and other valuable substances. The newly created bacterium has a smaller genetic code than does any natural free-living counterpart, with 531,000 DNA building blocks containing 473 genes. (Humans have more than 3 billion building blocks and more than 20,000 genes). But even this stripped-down organism is full of mystery. Scientists say they have little to no idea what a third of its genes actually do. "We're showing how complex life is, even in the simplest of organisms," researcher J. Craig Venter told reporters. "These findings are very humbling." Some of the mystery genes may be clues to discovering unknown fundamental processes of life, his colleague Clyde Hutchison III said in an interview. Both researchers, from the J. Craig Venter Institute in La Jolla, California, are among the authors of a paper on the project released Thursday by the journal Science. The DNA code, or genome, is contained in a brand-new bacterium dubbed JCVI-syn3.0. The genome is not some one-and-only minimal set of genes needed for life itself. For one thing, if the researchers had pared DNA from a different bacterium they would probably have ended up with a different set of genes. For another, the minimum genome an organism needs depends on the environment in which it lives. And the new genome includes genes that are not absolutely essential to life, because they help the bacterial populations grow fast enough to be practical for lab work. The genome is "as small as we can get it and still have an organism that is ... useful," Hutchison said. One goal of such work is to understand what each gene in a living cell does, which would lead to a deep understanding of how cells work, he said. With the new bacterium, "we're closer to that than we are for any other cell," he said. Another goal is to use such minimal-DNA microbes as a chassis for adding genes to make the organisms produce medicines, fuels and other substances for uses like nutrition and agriculture, said study co-author Daniel Gibson of Synthetic Genomics in La Jolla. The work began with a manmade version of a microbe that normally lives in sheep, called M. mycoides (my-KOY'-deez). It has about 900 genes. The scientists identified 428 nonessential genes, built their new genome without them, and showed that it was complete enough to let a bacterium survive. Experts not involved with the work were impressed. "I find this paper really ground breaking," said Jorg Stulke of the University of Goettingen in Germany, who is working on a similar project with a different bacterium. In an email, he said the researchers seem to have gotten at least very close to a minimum genome for M. mycoides. Farren Isaacs of Yale University called the work "an impressive tour de force," one that may begin to identify "a universe of minimal genomes."