Nicola J. Patron

Editing plant genomes with CRISPR/Cas9

CRISPR/Cas9 is a rapidly developing genome editing technology that has been successfully applied in many organisms, including model and crop plants. Cas9, an RNA-guided DNA endonuclease, can be targeted to specific genomic sequences by engineering a separately encoded guide RNA with which it forms a complex. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Remarkably, in some plant species, homozygous knockout mutants can be produced in a single generation. Together with other sequence-specific nucleases, CRISPR/Cas9 is a game-changing technology that is poised to revolutionise basic research and plant breeding.

Gene Replacement of Fructose-1,6-Bisphosphate Aldolase Supports the Hypothesis of a Single Photosynthetic Ancestor of Chromalveolates

ABSTRACT Plastids (photosynthetic organelles of plants and algae) are known to have spread between eukaryotic lineages by secondary endosymbiosis, that is, by the uptake of a eukaryotic alga by another eukaryote. But the number of times this has taken place is controversial. This is particularly so in the case of eukaryotes with plastids derived from red algae, which are numerous and diverse. Despite their diversity, it has been suggested that all these eukaryotes share a recent common ancestor and that their plastids originated in a single endosymbiosis, the so-called “chromalveolate hypothesis.” Here we describe a novel molecular character that supports the chromalveolate hypothesis. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic and Calvin cycle enzyme that exists as two nonhomologous types, class I and class II. Red algal plastid-targeted FBA is a class I enzyme related to homologues from plants and green algae, and it would be predicted that the plastid-targeted FBA from algae with red algal secondary endosymbionts should be related to this class I enzyme. However, we show that plastid-targeted FBA of heterokonts, cryptomonads, haptophytes, and dinoflagellates (all photosynthetic chromalveolates) are class II plastid-targeted enzymes, completely unlike those of red algal plastids. The chromalveolate enzymes form a strongly supported group in FBA phylogeny, and their common possession of this unexpected plastid characteristic provides new evidence for their close relationship and a common origin for their plastids.

Multiple Gene Phylogenies Support the Monophyly of Cryptomonad and Haptophyte Host Lineages

Cryptomonad algae acquired their plastids by the secondary endosymbiotic uptake of a eukaryotic red alga. Several other algal lineages acquired plastids through such an event [1], but cryptomonads are distinguished by the retention of a relic red algal nucleus, the nucleomorph [2]. The nucleomorph (and its absence in other lineages) can reveal a great deal about the process and history of endosymbiosis, but only if we know the relationship between cryptomonads and other algae, and this has been controversial. Several recent analyses have suggested a relationship between plastids of cryptomonads and some or all other red alga-containing lineages [3-6], but we must also know whether host nuclear genes mirror this relationship to determine the number of endosymbiotic events, and this has not been demonstrated. We have carried out an expressed sequence tag (EST) survey of the cryptomonad Guillardia theta. Phylogenetic analyses of 102 orthologous nucleus-encoded proteins (18,425 amino acid alignment positions) show a robust sister-group relationship between cryptomonads and the haptophyte algae, which also have a red secondary plastid. This relationship demonstrates that loss of nucleomorphs must have taken place in haptophytes independently of any other red alga-containing lineages and that the ancestor of both already contained a red algal endosymbiont.

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

BackgroundGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.ResultsPutative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.ConclusionETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.

The Altered Pattern of Amylose Accumulation in the Endosperm of Low-Amylose Barley Cultivars Is Attributable to a Single Mutant Allele of Granule-Bound Starch Synthase I with a Deletion in the 5′-Non-Coding Region

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5′-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxyancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.

The lys5 Mutations of Barley Reveal the Nature and Importance of Plastidial ADP-Glc Transporters for Starch Synthesis in Cereal Endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.

A Golden Gate Modular Cloning Toolbox for Plants

Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant Nicotiana benthamiana is discussed.

A Low-Starch Barley Mutant, Risø 16, Lacking the Cytosolic Small Subunit of ADP-Glucose Pyrophosphorylase, Reveals the Importance of the Cytosolic Isoform and the Identity of the Plastidial Small Subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis.

This work identifies an enzyme, named Like Sex Four2 (LSF2), that hydrolyzes the phosphate group from the C3-position of glucosyl residues of starch. The lsf2 mutation in Arabidopsis thaliana leads to modified starch with elevated levels of C3-bound phosphate. Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.