Ana Sofia Pires

Genome size analyses of Pucciniales reveal the largest fungal genomes.

Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production “on demand” and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N‐glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.

A novel plant α4-fucosyltransferase (Vaccinium myrtillus L.) synthesises the Lewisa adhesion determinant

We have partially characterised an α4‐fucosyltransferase (α4‐FucT) from Vaccinium myrtillus, which catalysed the biosynthesis of the Lewisa adhesion determinant. The enzyme was stable up to 50°C. The optimum pH was 7.0, both in the presence and in the absence of Mn2+. The enzyme was inhibited by Mn2+ and Co2+, and showed resistance towards inhibition with N‐ethylmaleimide. It transferred fucose to N‐acetylglucosamine in the type I Galβ3GlcNAc motif from oligosaccharides linked to a hydrophobic tail and glycoproteins (containing the type I motif). Sialylated oligosaccharides containing the type II Galβ4GlcNAc motif were not acceptors. The catalytic mechanism of the plant α4‐FucT possibly involves a His residue, and it must have arisen by convergent evolution relative to its mammalian counterparts.

EFFICIENT SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM LONG-TERM CELL SUSPENSION CULTURES OF MEDICAGO TRUNCATULA CV. JEMALONG

SummaryPlants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica—C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

Cell-line-dependent sorting of recombinant phytase in cell cultures of Medicago truncatula

Plant cell cultures as platforms for recombinant protein production are favoured over other systems because they combine the benefits of plants (low cost of production, low biosecurity risk, conserved post-translational modifications) with those of controlled cell cultures. However, many factors that affect the correct synthesis and accumulation of the recombinant product still need to be determined; in particular, the trafficking route of the recombinant proteins is poorly understood. Suspension cell cultures of Medicago truncatula Gaertn. have been shown to offer a viable and highly efficient system for the production of a model glycoprotein - phytase from the fungus Aspergillus niger Tiegh. The present study investigated subcellular protein sorting by immunogold detection of recombinant phytase with an electron microscope in four independent Medicago cell cultures expressing phytase. Two lines contained a C-terminal KDEL targeting signal for retention in the endoplasmic reticulum (ER), and the other two did not and were expected to travel through the secretory route; a high and low expressor were examined for each variant of the protein. A differential subcellular location of phytase was found in the four transgenic lines studied. These differences account not only for the version of the recombinant protein (secreted or retained in the ER), but also for the different expression levels.

Legitimacy and Implications of Reducing Colletotrichum kahawae to Subspecies in Plant Pathology

Colletotrichum kahawae Waller and Bridge is a highly aggressive and specialized fungal pathogen of coffee, causing the devastating Coffee Berry Disease (CBD), particularly at high altitudes. The disease arises from the unique ability of the pathogen to infect green developing coffee berries. This pathogen is currently confined to the African continent in all countries that grow Arabica coffee (Coffea arabica L.), leading to up to 80% yield losses, if no control measures are applied (Silva et al., 2006; Vossen and Walyaro, 2009; Hindorf and Omondi, 2011). For such huge economic impact, it is ranked as a quarantine pathogen and even as a biological weapon (Australia Group, 2014). Consequently, the pathogens potential dispersal to other Arabica coffee cultivation regions is greatly feared, particularly to those at high altitude also found in Latin America and Asia. Recently, this recognized species was brought down to a subspecific level (C. kahawae subsp. kahawae) based on molecular data (Weir et al., 2012), clustering together with a generalist and cosmopolitan group of Colletotrichum isolates unable to cause CBD (C. kahawae subsp. ciggaro). Since then a growing number of studies have reported the identification of C. kahawae in various hosts and regions of the world (Liu et al., 2013; Afanador-Kafuri et al., 2014; Mosca et al., 2014; Schena et al., 2014; Ismail et al., 2015; Garibaldi et al., 2016a,b; Perrone et al., 2016). Although these reports are referring to C. kahawae subsp. ciggaro, some of them could not distinguish the pathogen at the subspecific level, and this is leading to a wave of confusion of whether the long accepted species C. kahawae, the CBD pathogen, has escaped from Africa and extended its host range. Given the extreme impact that this situation may trigger and the subsequent biosecurity implications, there is a practical need to completely distinguish these pathogens taxonomically as to avoid the risk of misidentification, and caution should be taken on assigning/reassigning taxonomic ranking and nomenclature. Here we consider the evidences sustaining and contradicting the classification proposed by Weir et al. (2012), and discuss the risks and practical implications of changing the CBD pathogens species status in a plant pathology context.

Characterization of Medicago truncatula cell suspension cultures producing valuable recombinant proteins

Address: 1Plant Cell Biology Laboratory, Instituto de Tecnologia Quimica e Biologica – UNL. Av. Republica, Apartado 127, 2781-901 Oeiras, Portugal, 2Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Quimica e Biologica – UNL. Av. Republica, Apartado 127, 2781-901 Oeiras, Portugal and 3Department of Plant Biology, Centro de Investigaciones Biológicas, CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain * Corresponding author

Integrated approaches to studying Medicago truncatula genome structure and function and their applications in biotechnology

Plant genetic engineering has become an invaluable tool in plant research. Although plant transformation is a well-established technique, transgene expression is still unpredictable. Silencing may involve epigenetic modifications or nuclear and chromosomal localization of transgenes. In this way, understanding nuclear structure and organization is important not only for increasing our knowledge of fundamental aspects of the genome but also for taking the greatest advantage of inserting foreign genes and controlling their expression in biotechnological applications. Integrated approaches are clearly required in order to elucidate such complex processes. By combining the analysis of the physical position of transgenes with markers for epigenetic modifications in the plant genome we can better understand the factors affecting transgene expression levels and analyze the genomic environments of differentially expressed transgenes. Medicago truncatula Gaertn. has become a well-known model for the legume family and is used in studies ranging from nodulation to environmental stresses. More recently its use in biotechnology has been explored. In this report we describe the application of fluorescence in situ hybridization (FISH) to detect foreign DNA sequences and to determine the organization of the nucleolar organizer regions (NORs) genes in both metaphase chromosomes and interphase nuclei. We also studied chromatin distribution by immunodetection of epigenetic marks in M. truncatula interphase nuclei from tissue sections. We present evidence that M. truncatula is amenable to this kind of studies, which will in turn contribute to a better exploitation of biotechnology applications for this important plant family.