Monia Teresa Russo

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom

Summary Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo‐nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex‐related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein‐coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis‐related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient‐replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.

Natural variation of model mutant phenotypes in Ciona intestinalis.

Background The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. Methodology/Principal Findings Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. Conclusions/Significance Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene

We report the genetic transformation of the planktonic diatoms Pseudo-nitzschia arenysensis and Pseudo-nitzschia multistriata, members of the widely distributed and ecologically important genus Pseudo-nitzschia. P. arenysensis and P. multistriata present the classical size reduction/restitution life cycle and can reproduce sexually. Genetic transformation was achieved with the biolistic method, using the H4 gene promoter from P. multistriata to drive expression of exogenous genes. The transformation was first optimized introducing the Sh ble gene to confer resistance to the antibiotic zeocin. Integration of the transgene was confirmed by PCR and Southern blot analyses. Subsequently, we simultaneously transformed in P. arenysensis two plasmids, one encoding the β-glucuronidase (GUS) gene together with the plasmid carrying the Sh ble resistance gene, demonstrating the possibility of co-transformation. By transforming a gene encoding a fusion between the histone H4 and the green fluorescent protein (GFP), we demonstrated that fluorescent tagging is possible and that studies for protein localization are feasible. Importantly, we crossed P. arenysensis- and P. multistriata-transformed strains with a wild-type strain of opposite mating type and demonstrated that the transgene can be inherited in the F1 generation. The possibility to transform two diatom species for which genetic crosses are possible opens the way to a number of new approaches, including classical loss of function screens and the possibility to obtain different combinations of double transformants.

Expression of a single prominin homolog in the embryo of the model chordate Ciona intestinalis

Prominins are a family of pentaspan transmembrane glycoproteins, expressed in various types of cells, including stem and cancer stem cells in mammals. Prominin-1 is critical in generating and maintaining the structure of the photoreceptors in the eye since mutations in the PROM1 gene are associated with retinal and macular degeneration in human. In this study, we identified a single prominin homolog, Ci-prom1/2, in the model chordate the ascidian Ciona intestinalis and characterized Ci-prom1/2 expression profile in relation to photoreceptor differentiation during Ciona embryonic development. In situ hybridization experiments show Ci-prom1/2 transcripts localized in the developing central nervous system, predominantly in photoreceptor cell precursors as early as neurula stage and expression is maintained through larva stage in photoreceptor cells around the simple eye. We also isolated the regulatory region responsible for the specific spatio-temporal expression of the Ci-prom1/2 in photoreceptor cell lineage. Collectively, we report that Ci-prom1/2 is a novel molecular marker for ascidian photoreceptor cells and might represent a potential source to enlarge the knowledge about the function of prominin family in photoreceptor cell evolution and development.

Genome editing in diatoms: achievements and goals

Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.

Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, co-opted from a bacterial defense natural mechanism, is the cutting edge technology to carry out genome editing in a revolutionary fashion. It has been shown to work in many different model organisms, from human to microbes, including two diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana. Transforming P. tricornutum by bacterial conjugation, we have performed CRISPR/Cas9-based mutagenesis delivering the nuclease as an episome; this allowed for avoiding unwanted perturbations due to random integration in the genome and for excluding the Cas9 activity when it was no longer required, reducing the probability of obtaining off-target mutations, a major drawback of the technology. Since there are no reports on off-target occurrence at the genome level in microalgae, we performed whole-genome Illumina sequencing and found a number of different unspecific changes in both the wild type and mutant strains, while we did not observe any preferential mutation in the genomic regions in which off-targets were predicted. Our results confirm that the CRISPR/Cas9 technology can be efficiently applied to diatoms, showing that the choice of the conjugation method is advantageous for minimizing unwanted changes in the genome of P. tricornutum.