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A forward genetic screen in the marine planktonic diatom Pseudo-nitzschia multistriata

Final Report Summary - GYPSY (A forward genetic screen in the marine planktonic diatom Pseudo-nitzschia multistriata)

After being for more than a century the object of study of knowledgeable and passionate algologists, who described their astonishing forms and different physiological and ecological features, diatoms have been moving further in the spotlight because of their promising potential as sources of lipids and novel compounds for biotechnological application. Moreover, data from recent global oceanic expeditions remarked diatoms biodiversity, ubiquitous distribution and relevance to aquatic ecosystems.
Despite diatoms importance, little is known about the molecular mechanisms that underlie their ability to interact with biotic and abiotic components of the environment and to adapt to external changes. Most of the studies have been conducted on two species, Phaeodactylum tricornutum and Thalassiosira pseudonana, easily maintained in culture, which have provided a plethora of information about diatom biology, mainly through the exploration of their genomes and through functional studies.
The main idea behind the GyPSy project (Genetics in PSeudo-nitzschia) was to choose, among the estimated 100,000 diatom species, a diatom which could provide complementary information and additional advantages with respect to the two classical models. The species Pseudo-nitzschia multistriata was selected for three main reasons: 1) it is member of a widely distributed genus, of great relevance in marine ecosystems, 2) it is a toxic species, being able to produce the harmful neurotoxin domoic acid (DA), 3) its life cycle and population genetics have been well described. This last point was specifically important because controllable genetics, i.e. the possibility to set-up crosses between strains of opposite mating type and isolate consecutive generations, implied that classical approaches applied to genetic model organisms could be devised for diatoms too.
The project’s objectives were set to demonstrate that P. multistriata was a promising model species and to produce resources that would allow exploitation of this diatom to answer to basic and specific biological questions. Namely, the main objectives were : I) to consolidate P. multistriata as model species, optimising culturing conditions and genetic crosses; II) to sequence its genome; III) to optimise mutagenesis protocols; IV) to identify clones with altered production of fatty acids, oxylipins and domoic acid; V) to carry out a preliminary characterisation of the mutant lines investigating the pathways affected.
During the first year of the project, efforts have been devoted to obtain improvements in the cultivation methods and to begin crosses to establish a long-term pedigree. Over the course of four years, we have obtained six consecutive generations, recording a generation time of ca six months under standard growth conditions which can be made even shorter by adjusting growth parameters, mainly light intensity. We have also tested different combinations of antibiotics to establish axenic cultures. The main issue encountered concerning this first objective has been the failure of all the cryopreservation protocols tested. Currently, we cannot preserve a strain for longer than four-five years, but we can use genetic crosses to propagate a character of interest to the next generation.
For the genome project, we selected a second generation strain, obtained by the cross of two siblings, to produce the genome sequence. The strain was made axenic, genomic DNA was extracted from different batches of the same culture over two years and used to produce five different libraries for Illumina sequencing. 135 billion bp were sequenced in total. Different assemblies were produced, the final one comprised 1098 scaffolds for a genome of 59 Mb with N50 of 138 kb and a completeness of 89.11% (CEGMA analysis). Gene models were predicted with the support of RNA-seq data generated for a different project. Annotation was done using a recently developed pipeline called Annocript. In the last year, an intensive analysis of the genome data has yielded information on the amount of repeats, on conserved non-coding elements, on gene gains and losses, on gene families expansion and on the origin of genes. Comparison with the other diatom genomes has also been performed and has revealed that ca 20% of the P. multistriata genes are unique to this species (Basu, Patil et al, in preparation). Sequencing, assembly and gene models predictions were made in collaboration with the Genome Analysis Centre (TGAC, UK), gene annotation and downstream analyses were made at the Stazione Zoologica with personnel supported by GyPSy. A genome browser populated with several tracks for gene models, RNA-seq data, repeats, conserved sequences etc., is already available for internal use and will be made public upon publication of the genome paper.
We have optimized a protocol for ENU mutagenesis, and have used herbicide-resistance screenings to test the efficiency of the experiments. In addition, we have preliminary explored the use of flow cytometry in combination with vital stains for lipids to isolate mutants with increased production of lipids. In parallel to random mutagenesis, several attempts were made to introduce exogenous DNA in P. multistriata cells using the biolistic method. Exploiting the genome sequence, we cloned a P. multistriata promoter to drive expression of a gene conferring resistance to the antibiotic zeocin, transformed P. multistriata cells and obtained zeocin-resistant strains. Importantly, we also demonstrated that, using genetic crosses, the transgene can be carried over to the progeny (Sabatino et al., 2015). The possibility to transform a diatom species for which genetic crosses are possible opens the way to a number of new approaches, including alternative possibilities to generate mutants. Indeed, while screening for oxylipins mutants in the forward genetics screen proved unfeasible for the elevated costs, we are now able to use reverse genetics approaches and are targeting key genes in the oxylipins pathway with RNA interference.
An important issue with many Pseudo-nitzschia species is their ability to produce DA, a neurotoxin that can cause amnesic shellfish poisoning: humans, marine mammals and birds that consume contaminated shellfish can develop severe neurological symptoms leading to death in the worst cases. The mechanisms of DA production to date are unknown. To obtain insights into the metabolic pathways leading to DA production, we again exploited the genome and the transcriptome sequences to guide experiments in the lab: we compared the transcriptome of the toxic P. multistriata with the transcriptome of the non-toxic P. arenysensis to produce a list of candidate genes involved in DA production for further experimental validations via qPCR (Adelfi et al., 2014, Di Dato et al. 2015). Similarly, relying on the reference genome sequence, we obtained independent funds to re-sequence the genomes of toxic and non-toxic P. multistriata strains to further restrict the list of candidate genes putatively responsible for the toxicity.
In summary, with the support of this project important resources have been generated that are currently allowing to use P. multistriata as a model species to investigate specific questions linked to the species biology. More broadly, the availability of the genome sequence is allowing a number of comparisons with other diatoms and with other taxonomic groups for the identification of unique and conserved functions. As an example, we have defined the molecular toolkit that diatoms possess to perform meiosis, the process at the base of gamete production and sexual reproduction (Patil et al., in revision). Parallel projects aimed at identifying the sex locus in P. multistriata and in defining the complex gene expression changes occurring during sexual reproduction are also profiting from the resources generated thanks to GyPSy.
The data produced within this project represent a relevant contribution for understanding diatoms evolution and ecology, and are specifically important for comprehending the dynamics of toxic blooms, which constitute a major problem for aquaculture. Finally, the possibility apply genetic engineering to P. multistriata will be important for potential industrial applications, providing the opportunity to ameliorate strain properties or to boost production of specific compounds.