Synthetic biology in chloroplasts

Dr Anil Day, University of Manchester

Dr Anil Day, University of Manchester

This week we spoke to Dr. Anil Day, a synthetic biologist at the University of Manchester who has developed an impressive array of tools and techniques to transform chloroplast genomes.

 

Could you begin by giving our readers a brief overview of synthetic biology?

Synthetic biology involves the application of engineering principles to biological systems. One approach to understanding a biological system is to break it down into smaller parts, which can be used to design new properties. These redesigned pieces can be reassembled into a new system, tested experimentally, and refined in an iterative process. Synthetic biology projects that are underway in our lab include designing plastids such as chloroplasts with new metabolic functions, and in the longer term the design and assembly of synthetic chloroplast genomes.

 

Anil Day examines transformed plants

Dr. Anil Day examines a cabinet of transformed plants. Credit: Dr. Anil Day.

Why do you use chloroplasts for synthetic biology systems?

Chloroplasts have a relatively small genome, coding for about 100 genes. Importantly, exogenous (foreign) genes coding for new functions can be precisely introduced into the chloroplast genome. All of the plastids within a plant contain the same genome so, once established, the user-designed reprogrammed plastids will be present throughout the plant. Chloroplasts can also produce very high levels of protein; researchers have achieved expression levels where over 70% of the total soluble protein in the leaves is the engineered protein. Expression in tomato fruit is also possible.

Multiple genes can be introduced into chloroplasts and expressed coordinately, allowing the metabolic engineering of more complex processes. The upper size limit for insertions is not known but is likely to be above the 50,000 nucleotide insertion achieved to date. Furthermore, chloroplasts and other plastids are important metabolic hubs and contain a wide variety of chemical substrates useful for metabolic engineering.

Plastids in plants

Plants have several types of plastids, including green photosynthetic chloroplasts, pigment-containing chromoplasts, and starch-containing amyloplasts. Credit: Dr. Anil Day.

 

Could you describe the current state of our ability to engineer chloroplasts?

Chloroplast engineering is routine in many labs around the globe. Although there are multiple chloroplasts in every cell, the process of converting all the chloroplasts to a single population of engineered genomes is not an issue. Most researchers use the tobacco plant because it is easily transformed, but other crops are amenable to transformation, including oilseed rape, soybean, tomato, and potato (cereals such as rice and wheat are more problematic). There has been progress with developing the inducible expression of exogenous genes in chloroplasts too.

 

What challenges/differences do you face when transforming chloroplast genomes when compared to the nuclear genome?

Typical genetic modification of the DNA in the nucleus is performed by introducing exogenous genes in T-DNA. T-DNA is transferred to the plant using the bacterium Agrobacterium tumefaciens, which is an efficient process, but the T-DNA integrates ‘randomly’ at many sites within chromosomes and different lines can have variable expression levels due to positional effects and gene silencing.

A. tumefaciens-mediated gene delivery systems do not work for chloroplast transformation. Most chloroplast transformation labs introduce genes into plastids by blasting cells with gold or tungsten particles coated with DNA. Because chloroplast genomes are present in multiple copies per cell, the process of converting all resident chloroplasts to the transgenic genome requires a continued period of selection. This means that the isolation of chloroplast transformants can take slightly longer than nuclear transformation. In our lab, we speed up this process by using restoration of photosynthesis to select chloroplasts with exogenous genes. Once plants with a uniform population of transgenic plastid genomes have been isolated, the transgenes are stable and inherited through the maternal line.

For the novice, I would say nuclear transformation using A. tumefaciens is easier to accomplish than chloroplast transformation.

 

Edited chloroplasts

A tobacco plant containing leaf areas with edited (pale green) and normal (darker green) chloroplasts. Credit: Dr. Anil Day.

Last year you reported that chloroplasts degrade in mature sperm cells just prior to fertilization. Could you elaborate on how this might be utilized in future crop breeding?

Chloroplasts are inherited from the female parent in wheat. This is useful because it restricts the pollen-mediated spread of chloroplast-localized transgenes into the environment. Previously, no-one had studied the mechanism of maternal chloroplast inheritance in wheat using modern cell biology tools. With our collaborators Lucia PrimavesiHuixia Wu, and Huw Jones at Rothamsted Research, we developed an efficient method to observe small non-green plastids in wheat pollen in real time. We found that the plastids were destroyed during the maturation of sperm cells, which explained the absence of paternal plastids in the offspring.

This discovery has applications in crop breeding. Anther culture is a powerful technique where new homozygous plants can be produced by doubling the chromosome numbers of haploid plants regenerated from pollen. This technique has been challenging in cereals, as chloroplast degradation in pollen leads to a high percentage of albino plants (in some cases 100% albinos). Understanding how to prevent the destruction of plastids in pollen sperm cells will improve this technique in cereals, which could speed up crop breeding in the future.

 

Selection of transformed plants

Transformed plantlets are selected by their ability to survive on a herbicide-containing agar plate, and can then be grown up into mature plants. Credit: Dr. Anil Day.

 What sorts of processes have you successfully transformed into chloroplasts, and what kinds of results have you achieved?

We have expressed a variety of exogenous genes in chloroplasts, from those conferring resistance to herbicides to vaccine epitopes and pharmaceutical proteins:

  • Plants expressing the bar gene in chloroplasts were resistant to the herbicide glufosinate (also known as phosphinothricin).
  • A chloroplast-expressed viral epitope was used to identify samples of human blood infected with the hepatitis C virus.
  • Human transforming growth factor 3 (hTGFβ3), a potential wound healing drug, accumulated to high concentrations in chloroplasts, and could be processed to a pure active form resembling clinical grade hTGFβ3.
  • In collaboration with Ray Dixon, Cheng Qi, and Mandy Dowson-Day at the John Innes Centre, we investigated the feasibility of introducing nitrogen-fixing genes into chloroplasts. This work was initiated in a unicellular green alga with the bacterial nifH gene.

 

What is the cutting edge of chloroplast transformation research?

Chloroplast genes are important for plant growth and development but they are difficult to improve by conventional breeding methods. We recently developed a method to edit plastid genomes, which allows beneficial single point mutations to be introduced into chloroplast genes. This is important because the resulting plants have an identical genome to the original cultivar apart the single base substitution, potentially leading to a new class of biotech crop.

2015 Plant Science Round Up

Following on from last week’s post, Now That’s What I Call Plant Science 2015, we bring you a year in Plant Science!

January

Arabidopsis

Image credit: Jean Weber. Used under license CC BY 2.0.

The year began with a surprising paper that turned our understanding of the phytohormone auxin on its head. Researchers in China and the USA created Arabidopsis knockout mutants of AUXIN BINDING PROTEIN 1 (ABP1), expecting them to fail to respond to auxin and have developmental defects, as previously seen in the abp1-1 knockdown mutant. Instead, these plants were indistinguishable from wild type plants, leading the authors to conclude that ABP1 is not required for auxin signaling or Arabidopsis development as previously believed.

Read the paper in PNAS: Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.

A paper later in the year from the same authors found that the embryonic lethality of the abp1-1 mutant is actually caused by the off-target linked deletion of the adjacent BSM gene.

Read this paper in Nature Plants: Embryonic lethality of Arabidopsis abp1-1 is caused by deletion of the adjacent BSM gene.

The tale of ABP1 was examined in more detail on the GARNet blog, Weeding the Gems, which concluded: “In many ways this story is an excellent example of how science should work, where claims are independently tested to ensure that earlier experiments have been conducted or interpreted correctly.” Click here to read more.

 

February

A clever experiment from Germany led to a significant breakthrough in crop protection from insect pests.

When double-stranded RNA (dsRNA) is present within a eukaryotic cell, it is cleaved by the Dicer enzyme to form short interfering RNAs. These can bind to complementary RNA within a cell to target it for destruction, thus silencing the corresponding gene expression. This process is known as RNA interference (RNAi).

RNAi has previously been used to tackle insect herbivory by expressing insect-specific dsRNA in plants; however the protection has previously been incomplete. In this new study, published in Science, researchers produced dsRNA within chloroplasts, which do not have RNAi machinery. When dsRNA is expressed in the cytoplasm, the plant’s own Dicer enzyme breaks most of it down. When expressed in the chloroplasts, the dsRNA remained intact when eaten by insects, which proved much more effective at killing these pests.

Read the paper here: Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.

 

March

Another crop protection study followed in March, when researchers in China cloned the genetic locus in rice that confers broad-spectrum resistance to planthoppers – insect pests that cause the loss of billions of dollars of crops per year. Three lectin receptor kinase genes were found in rice cultivars from the Philippines, which enable plants to survive an infestation of insects. When cloned into a susceptible rice cultivar, these genes conferred resistance to two different planthopper species.

Understanding the genetic basis of resistance is very important as marker-assisted breeding and selection could be used to develop resistant rice varieties, and potentially utilized in other species of cereal.

Read the paper in Nature Biotechnology: A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice

 

April

A European collaboration led to the development of 3DCellAtlas, a computational approach that semi-automatically identifies cell types in a developing 3D organ without the need for transgenic lineage markers. This program will enable the interpretation of dynamic organ growth and the spatial and temporal context of developmental cell divisions that produce the resultant plant. It could be integrated with growth in different conditions or with developmental mutants to examine exactly how these processes affect growth in 3D.

3DCellAtlas

Image credit: Montenegro-Johnson et al., 2015. Digital Single-Cell Analysis of Plant Organ Development Using 3DCellAtlas. The Plant Cell, vol. 27 no. 4, 1018–1033.

 

May

A special issue of the Plant Biotechnology Journal was published in May, focusing on the amazing advances in molecular farming. While the entire issue is worth delving into, we were particularly intrigued by the review on moss-made pharmaceuticals, which outlines the rapid progress made in the field.

The model moss Physcomitrella patens has rapidly become one of the organisms of choice in biotechnology, with a fully sequenced genome and an outstanding toolbox for genome-engineering. The authors describe how moss-made pharmaceuticals can easily be produced while remaining remarkably more stable from batch to batch than cultured animal cells. The system is easily scalable, making their production highly cost effective, and safe. The first moss-made pharmaceuticals are currently in clinical trials, so keep an eye out for much more from this field over the next few years.

Read the review: Moss-made pharmaceuticals: from bench to bedside.

 

June

In June, US researchers discovered a new role for chloroplast stromules, protrusions that extend from the surface of all plastid types. The function of stromules has been difficult to determine, but this research, published in Developmental Cell, suggests that they may provide a mechanism by which plastid signals are conveyed to the nucleus. The paper shows that chloroplast stromules are induced by defense responses such as programmed cell death signaling, and that the stromules extend to form dynamic connections with the nucleus. The stromules may therefore aid in the amplification and/or transport of immune response signals into the nucleus.

Read the paper: Chloroplast Stromules Function during Innate Immunity.

 

July

Extracellular self-DNA

Image credit: Veresoglou et al., 2015. Self-DNA: a blessing in disguise? New Phytologist, vol. 207, no. 3, 488–490.

In late 2014 and early 2015, Italian researchers published a set of articles showing that extracellular self-DNA, DNA from conspecifics, could inhibit the growth of organisms from a wide range of taxa, including plants, bacteria, fungi and animals. Conversely, these organisms were not affected by extracellular DNA from other unrelated species.

In July, New Phytologist published a letter offering an interpretation of the data as it relates to plants. Plants could interpret extracellular self-DNA as an indicator of intraspecific competition (which seeds could use as a cue to remain dormant) or of a hostile environment that has already caused the death of conspecifics, signaling them to ramp up their pre-emptive immune response to increase survival after neighbors have been damaged or killed. There are still a lot of mechanisms and ecological effects to be investigated in this new field, but this letter suggests several interesting avenues to investigate.

Read the article: Self-DNA: a blessing in disguise?

Original research papers in New Phytologist:

Inhibitory and toxic effects of extracellular self-DNA in litter: a mechanism for negative plant–soil feedbacks?

Inhibitory effects of extracellular self-DNA: a general biological process?

 

August

A US study in August revealed a surprising degree of conservation in gene expression patterns across a wide range of plant taxa during root development. This was particularly interesting because the spikemoss Selaginella was shown to use many of the same genes as the evolutionarily distant angiosperms, despite the fossil record suggesting that roots evolved independently in these two lineages. Perhaps roots in these two groups evolved by independently recruiting the same developmental program, or perhaps by elaborating on a previously unknown proto-root that existed in the common ancestor of vascular plants.

Read the paper in The Plant Cell: Conserved Gene Expression Programs in Developing Roots from Diverse Plants.

 

September

Salt stress can significantly reduce the growth and yield of plants. Researchers in Germany identified two components of the cellulose synthase complex that directly interact with the microtubules and promote their dynamics, which interestingly were highly produced during salt stress conditions. During salt stress, cellulose microtubules depolymerize, however the newly discovered compounds, known as Companions of Cellulose Synthase, promote the reassembly of the microtubule to allow cellulose synthesis to continue.

Read the paper in Cell: A Mechanism for Sustained Cellulose Synthesis during Salt Stress

 

October

Throughout the year the GM debate in Europe reached several important milestones. In January the European Union (EU) changed its rules, giving individual countries more flexibility to decide for themselves whether or not to plant GM crops. In February, the UK Science and Technology Committee report stated that EU regulations preventing GM crops are not fit for purpose, and that they should be replaced with a trait-based system.

In October, EU member states revealed their stances on GM crops, with over half of Europe opting out of growing GM crops. Germany was the largest country to opt out of growing GM. The full list can be viewed here: Restrictions of geographical scope of GMO.

Read the news articles here:

EU changes rules on GM crop cultivation – January 2015

EU regulation on GM Organisms not ‘fit for purpose’ – February 2015

Half of Europe opts out of new GM crop scheme – October 2015

 

November

A collaboration between South African and UK scientists revealed how plants can use their circadian clock to pre-emptively boost their immune resistance at dawn, when fungal infection is most likely. Plants tend to decrease in susceptibility at dawn, but those with dysfunctional circadian clocks remained highly susceptible throughout the day. The research also showed that jasmonate signaling plays a crucial role in the circadian timing of resistance.

Read the article in The Plant Journal: Jasmonate signalling drives time-of-day differences in susceptibility of Arabidopsis to the fungal pathogen Botrytis cinerea.

 

December

Single nucleotide exon

Image credit: Guo & Liu., 2015. A single-nucleotide exon found in Arabidopsis. Scientific Reports, 5:18087.

Researchers in China published the surprising finding that a single-nucleotide exon exists in the APC11 gene in Arabidopsis. This is the smallest exon ever to be discovered before. The team used an elegant set of APC11-GFP constructs to show that intron splicing around the single-nucleotide exon is effective in both Arabidopsis and rice. This finding has implications for future genome annotations, which might reveal many more single-nucleotide exons.

Read the paper in Scientific Reports: A single-nucleotide exon found in Arabidopsis.

 

What a wonderful year of science! What new knowledge will 2016 bring?