Solanum pennellii (LA5240) backcross inbred lines (BILs) for high resolution mapping in tomato.

Wild species are an invaluable source of new traits for crop improvement. Over the years, the tomato community bred cultivated lines that carry introgressions from different species of the tomato tribe to facilitate trait discovery and mapping. The next phase in such projects is to find the genes that drive the identified phenotypes. This can be achieved by genotyping a few thousand individuals resulting in fine mapping that can potentially identify the causative gene. To couple trait discovery and fine mapping, we are presenting large, recombination-rich, Backcross Inbred Line (BIL) populations involving an unexplored accession of the wild, green-fruited species Solanum pennellii (LA5240; the 'Lost' Accession) with two modern tomato inbreds: LEA, determinate, and TOP, indeterminate. The LEA and TOP BILs are in BC2F6-8 generation and include 1400 and 500 lines, respectively. The BILs were genotyped with 5000 SPET markers, showing that in the euchromatic regions there was one recombinant every 17-18 Kb while in the heterochromatin a recombinant every 600-700 Kb (TOP and LEA respectively). To gain perspective on the topography of recombination we compared five independent members of the Self-pruning gene family with their respective neighboring genes; based on PCR markers, in all cases we found recombinants. Further mapping analysis of two known morphological mutations that segregated in the BILs (self-pruning and hairless) showed that the maximal delimited intervals were 73 Kb and 210 Kb, respectively, and included the known causative genes. The 'Lost'_BILs provide a solid framework to study traits derived from a drought-tolerant wild tomato.

Generation and physiological characterization of genome-edited Nicotiana benthamiana plants containing zeaxanthin as the only leaf xanthophyll.

MAIN CONCLUSION: Simultaneous genome editing of the two homeologous LCYe and ZEP genes of Nicotiana benthamiana results in plants in which all xanthophylls are replaced by zeaxanthin. Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC-MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.

Analysis of >3400 worldwide eggplant accessions reveals two independent domestication events and multiple migration-diversification routes.

Eggplant (Solanum melongena) is an important Solanaceous crop, widely cultivated and consumed in Asia, the Mediterranean basin, and Southeast Europe. Its domestication centers and migration and diversification routes are still a matter of debate. We report the largest georeferenced and genotyped collection to this date for eggplant and its wild relatives, consisting of 3499 accessions from seven worldwide genebanks, originating from 105 countries in five continents. The combination of genotypic and passport data points to the existence of at least two main centers of domestication, in Southeast Asia and the Indian subcontinent, with limited genetic exchange between them. The wild and weedy eggplant ancestor S. insanum shows admixture with domesticated S. melongena, similar to what was described for other fruit-bearing Solanaceous crops such as tomato and pepper and their wild ancestors. After domestication, migration and admixture of eggplant populations from different regions have been less conspicuous with respect to tomato and pepper, thus better preserving 'local' phenotypic characteristics. The data allowed the identification of misclassified and putatively duplicated accessions, facilitating genebank management. All the genetic, phenotypic, and passport data have been deposited in the Open Access G2P-SOL database, and constitute an invaluable resource for understanding the domestication, migration and diversification of this cosmopolitan vegetable.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

In Vivo Functional Properties of Dairy Bacteria.

This literature review aimed to collect investigations on the in vivo evidence for bacteria associated with fermented dairy foods to behave as probiotics with beneficial effects in the prevention and treatment of various diseases. All main bacterial groups commonly present in high numbers in fermented milks or cheeses were taken into account, namely starter lactic acid bacteria (SLAB) Lactobacillus delbrueckii subsp. bulgaricus and lactis, L. helveticus, Lactococcus lactis, Streptococcus thermophilus, non-starter LAB (NSLAB) Lacticaseibacillus spp., Lactiplantibacillus plantarum, dairy propionibacteria, and other less frequently encountered species. Only studies regarding strains of proven dairy origin were considered. Studies in animal models and clinical studies showed that dairy bacteria ameliorate symptoms of inflammatory bowel disease (IBD), mucositis, metabolic syndrome, aging and oxidative stress, cancer, bone diseases, atopic dermatitis, allergies, infections and damage caused by pollutants, mild stress, and depression. Immunomodulation and changes in the intestinal microbiota were the mechanisms most often involved in the observed effects. The results of the studies considered indicated that milk and dairy products are a rich source of beneficial bacteria that should be further exploited to the advantage of human and animal health.

Cross-sectional study of hepatitis E virus (HEV) circulation in Italian pig farms.

Foodborne transmission is considered the main way of spreading zoonotic hepatitis E virus (HEV) infection in Europe. In recent years, the human cases of hepatitis E in subjects without history of travel in endemic areas have raised, suggesting that domestic HEV transmission is increasing. Pork products with or without liver, are often indicated as the source of many human foodborne HEV cases as well as small outbreaks. Pigs are recognized as the main reservoir of the zoonotic HEV-3 genotype, the most frequently detected in human cases in the EU. In the absence of a harmonized surveillance of HEV circulation, data on prevalence are heterogeneous but confirm a widespread circulation of HEV-3 in pig herds across EU. HEV-3 can pass through the food chain from farm to fork when infected animals are slaughtered. In Italy, several studies reported the circulation of HEV-3 in pig farms, but results are heterogeneous due to different methodologies applied. In the present study, we performed a survey over 51 pig herds belonging to three main types of farms: breeding, fattening and farrow-to-finish. HEV-RNA was analyzed by broad range Real-time RT-PCR on 20 samples for each farm, obtained by pooling together feces from 10 individuals. Overall, HEV RNA was confirmed on 150 fecal pooled samples out of 1,032 (14.5%). At least one positive pooled sample was detected from 18 farms out of 51 tested (35.3%). By lowering the number of infected pigs at primary production, the risk of HEV-3 entering into the food chain can be reduced. Hence, information on HEV circulation in herds is highly relevant for choosing preventive measures and deserves development of a monitoring program and further investigations.

Heterologous expression of Bixa orellana cleavage dioxygenase 4-3 drives crocin but not bixin biosynthesis.

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, ß-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues.

Bacteriophage Therapy to Reduce Colonization of Campylobacter jejuni in Broiler Chickens before Slaughter.

Campylobacteriosis is the most commonly reported gastrointestinal disease in humans. Campybacter jejuni is the main cause of the infection, and bacterial colonization in broiler chickens is widespread and difficult to prevent, leading to high risk of occurrence in broiler meat. Phage therapy represents an alternative strategy to control Campylobacter in poultry. The aim of this work was to assess the efficacy of two field-isolated bacteriophages against experimental infections with an anti-microbial resistant (AMR) Campylobacter jejuni strain. A two-step phage application was tested according to a specific combination between chickens' rearing time and specific multiplicities of infections (MOIs), in order to reduce the Campylobacter load in the animals at slaughtering and to limit the development of phage-resistant mutants. In particular, 75 broilers were divided into three groups (A, B and C), and phages were administered to animals of groups B and C at day 38 (Φ 16-izsam) and 39 (Φ 7-izsam) at MOI 0.1 (group B) and 1 (group C). All broilers were euthanized at day 40, and Campylobacter jejuni was enumerated in cecal contents. Reductions in Campylobacter counts were statistically significant in both group B (1 log10 colony forming units (cfu)/gram (gr)) and group C (2 log10 cfu/gr), compared to the control group. Our findings provide evidence about the ability of phage therapy to reduce the Campylobacter load in poultry before slaughtering, also associated with anti-microbial resistance pattern.

Characterization and In Vitro Efficacy against Listeria monocytogenes of a Newly Isolated Bacteriophage, ɸIZSAM-1.

Listeria monocytogenes is a bacterial pathogen responsible of listeriosis, a disease that in humans is often related to the contamination of ready-to-eat foods. Phages are candidate biodecontaminants of pathogenic bacteria thanks to their ability to lyse prokaryotes while being safe for eukaryotic cells. In this study, ɸIZSAM-1 was isolated from the drain-waters of an Italian blue cheese plant and showed lytic activity against antimicrobial resistant Listeria monocytogenes strains. This phage was subjected to purification and in vitro efficacy tests. The results showed that at multiplicities of infection (MOIs) ≤ 1, phages were able to keep Listeria monocytogenes at low optical density values up to 8 h, with bacterial counts ranging from 1.02 to 3.96 log10 units lower than the control. Besides, ɸIZSAM-1 was further characterized, showing 25 principal proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis profile) and a genome of approximately 50 kilo base pairs. Moreover, this study describes a new approach to phage isolation for applications in Listeriamonocytogenes biocontrol in food production. In particular, the authors believe that the selection of phages from the same environments where pathogens live could represent a new approach to successfully integrating the control measures in an innovative, cost effective, safe and environmentally friendly way.

Identification of Beneficial Microbial Consortia and Bioactive Compounds with Potential as Plant Biostimulants for a Sustainable Agriculture.

A growing body of evidence demonstrates the potential of various microbes to enhance plant productivity in cropping systems although their successful field application may be impaired by several biotic and abiotic constraints. In the present work, we aimed at developing multifunctional synthetic microbial consortia to be used in combination with suitable bioactive compounds for improving crop yield and quality. Plant growth-promoting microorganisms (PGPMs) with different functional attributes were identified by a bottom-up approach. A comprehensive literature survey on PGPMs associated with maize, wheat, potato and tomato, and on commercial formulations, was conducted by examining peer-reviewed scientific publications and results from relevant European projects. Metagenome fragment recruitments on genomes of potential PGPMs represented in databases were also performed to help identify plant growth-promoting (PGP) strains. Following evidence of their ability to coexist, isolated PGPMs were synthetically assembled into three different microbial consortia. Additionally, the effects of bioactive compounds on the growth of individually PGPMs were tested in starvation conditions. The different combination products based on microbial and non-microbial biostimulants (BS) appear worth considering for greenhouse and open field trials to select those potentially adoptable in sustainable agriculture.

The role of staff and contaminated environmental surfaces in spreading of norovirus infection in a long-term health care facility in Italy.

Some residents and people from the staff of a geriatric health care facility in Teramo province, developed acute gastroenteritis from March 8th to March 21st 2017. A prompt epidemiological investigation was conducted to identify the etiological agent, the trace back the potential ways of transmission and control the infection. Information on the outbreak was collected through an epidemiological questionnaire. Faecal samples from all human cases (n = 50) and swabs from environmental surfaces were collected and analysed by RT-PCR for the presence of Norovirus (NoV). Among faecal samples, 34 out of 50 were positive for NoV with no other pathogen detected. In particular, 2 (2/34) were positive to NoV genogroup I (GI), 31 (31/34) to NoV genogroup II (GII), and one sample (1/34) was positive to both NoV GI and GII. Moreover, faecal samples of people from the canteen (n = 8) were also tested resulting negative to NoV detection. Norovirus was also detected in 28 of the 122 swabs from environmental surfaces collected. Among the positive samples, 12 NoV strains were subtyped as NoV GII.4 Sydney_2012 variant. Person-to-person close contact and contaminated environmental surfaces were the probable transmission route among the people of the health care facility. The members of the staff were considered to play an important role in transmission of NoV. A proper disinfection procedure applied during the outbreak could have been critically important to limit the dissemination of the viral infection.

Whole Genome Sequencing Characterization of HEV3-e and HEV3-f Subtypes among the Wild Boar Population in the Abruzzo Region, Italy: First Report.

Hepatitis E virus (HEV) is an emergent zoonotic pathogen, causing worldwide acute and chronic hepatitis in humans. HEV comprises eight genotypes and several subtypes. HEV genotypes 3 and 4 (HEV3 and HEV4) are zoonotic. In Italy, the most part of HEV infections (80%) is due to autochthonous HEV3 circulation of the virus, and the key role played by wild animals is generally accepted. Abruzzo is an Italian region officially considered an HEV "hot spot", with subtype HEV3-c being up to now the only one reported among wild boars. During the year 2018-2019, a group of wild boars in Abruzzo were screened for HEV; positive RNA liver samples were subjected to HEV characterization by using the whole genome sequencing (WGS) approach methodology. This represents the first report about the detection of HEV-3 subtypes e and f in the wild boar population in this area. Since in Italy human infections from HEV 3-e and f have been associated with pork meat consumption, our findings deserve more in-depth analysis with the aim of evaluating any potential correlation between wild animals, the pork chain production and HEV human infections.

Campylobacter bacteriophage DA10: an excised temperate bacteriophage targeted by CRISPR-cas.

BACKGROUND: Lytic bacteriophages that infect Campylobacter spp. have been utilized to develop therapeutic/decontamination techniques. However, the association of Campylobacter spp. and bacteriophages has been the focus of several strands of research aimed at understanding the complex relationships that have developed between predators and prey over evolutionary time. The activities of endogenous temperate bacteriophages have been used to evaluate genomic rearrangements and differential protein expression in host cells, and mechanisms of resistance to bacteriophage infection in campylobacters such as phase variation and CRISPR-mediated immunity. RESULTS: Temperate bacteriophage DA10 represents a novel excised and infective virus capable of replication in a restricted set of C. jejuni and C. coli hosts. Whole genome sequencing reveals that DA10 (35,379 bp) forms part of a novel group of temperate bacteriophages that have limited distribution among database host genome sequences. Analysis of potential host genomes reveals a robust response against DA10 and DA10-like bacteriophages is driven by CRISPR-mediated immunity with 75% of DA10 ORFs represented as ~ 30 bp spacer sequences in numerous Campylobacter Type II-C CRISPR arrays. Several DA10-like homologues have been identified in a small sub-set of C. jejuni and C. coli genome sequences (ranging from near complete integrated prophage sequences to fragments recognisable in the sequence read archive). CONCLUSIONS: A complete intact DA10-like prophage in C. jejuni CJ677CC520 provides evidence that the associations between host and DA10-like bacteriophages are long-standing in evolutionary timescales. Extensive nucleotide substitution and loss can be observed in the integrated DA10-like prophage of CJ677CC520 compared to other relatives as observed through pairwise genome comparisons. Examining factors that have limited the population expansion of the prophage, while others appear to have thrived and prospered (Mu-like, CJIE-like, and lytic Campylobacter bacteriophages) will assist in identifying the underlying evolutionary processes in the natural environment.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.

Single Primer Enrichment Technology (SPET) for High-Throughput Genotyping in Tomato and Eggplant Germplasm.

Single primer enrichment technology (SPET) is a new, robust, and customizable solution for targeted genotyping. Unlike genotyping by sequencing (GBS), and like DNA chips, SPET is a targeted genotyping technology, relying on the sequencing of a region flanking a primer. Its reliance on single primers, rather than on primer pairs, greatly simplifies panel design, and allows higher levels of multiplexing than PCR-based genotyping. Thanks to the sequencing of the regions surrounding the target SNP, SPET allows the discovery of thousands of closely linked, novel SNPs. In order to assess the potential of SPET for high-throughput genotyping in plants, a panel comprising 5k target SNPs, designed both on coding regions and introns/UTRs, was developed for tomato and eggplant. Genotyping of two panels composed of 400 tomato and 422 eggplant accessions, comprising both domesticated material and wild relatives, generated a total of 12,002 and 30,731 high confidence SNPs, respectively, which comprised both target and novel SNPs in an approximate ratio of 1:1.6, and 1:5.5 in tomato and eggplant, respectively. The vast majority of the markers was transferrable to related species that diverged up to 3.4 million years ago (Solanum pennellii for tomato and S. macrocarpon for eggplant). Maximum Likelihood phylogenetic trees and PCA outputs obtained from the whole dataset highlighted genetic relationships among accessions and species which were congruent with what was previously reported in literature. Better discrimination among domesticated accessions was achieved by using the target SNPs, while better discrimination among wild species was achieved using the whole SNP dataset. Our results reveal that SPET genotyping is a robust, high-throughput technology for genetic fingerprinting, with a high degree of cross-transferability between crops and their cultivated and wild relatives, and allows identification of duplicates and mislabeled accessions in genebanks.

A chromosome-anchored eggplant genome sequence reveals key events in Solanaceae evolution.

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

Pivotal Roles of Cryptochromes 1a and 2 in Tomato Development and Physiology.

Cryptochromes are flavin-containing blue/UVA light photoreceptors that regulate various plant light-induced physiological processes. In Arabidopsis (Arabidopsis thaliana), cryptochromes mediate de-etiolation, photoperiodic control of flowering, entrainment of the circadian clock, cotyledon opening and expansion, anthocyanin accumulation, and root growth. In tomato (Solanum lycopersicum), cryptochromes are encoded by a multigene family, comprising CRY1a, CRY1b, CRY2, and CRY3 We have previously reported the phenotypes of tomato cry1a mutants and CRY2 overexpressing plants. Here, we report the isolation by targeting induced local lesions in genomes, of a tomato cry2 knock-out mutant, its introgression in the indeterminate Moneymaker background, and the phenotypes of cry1a/cry2 single and double mutants. The cry1a/cry2 mutant showed phenotypes similar to its Arabidopsis counterpart (long hypocotyls in white and blue light), but also several additional features such as increased seed weight and internode length, enhanced hypocotyl length in red light, inhibited primary root growth under different light conditions, anticipation of flowering under long-day conditions, and alteration of the phase of circadian leaf movements. Both cry1a and cry2 control the levels of photosynthetic pigments in leaves, but cry2 has a predominant role in fruit pigmentation. Metabolites of the sterol, tocopherol, quinone, and sugar classes are differentially accumulated in cry1a and cry2 leaves and fruits. These results demonstrate a pivotal role of cryptochromes in controlling tomato development and physiology. The manipulation of these photoreceptors represents a powerful tool to influence important agronomic traits such as flowering time and fruit quality.

Candidate Enzymes for Saffron Crocin Biosynthesis Are Localized in Multiple Cellular Compartments.

Saffron is the dried stigmas of Crocus sativus and is the most expensive spice in the world. Its red color is due to crocins, which are apocarotenoid glycosides that accumulate in the vacuole to a level up to 10% of the stigma dry weight. Previously, we characterized the first dedicated enzyme in the crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) genes expressed in C. sativus stigmas. Heterologous expression in Escherichia coli showed that only one of corresponding proteins (CsALDH3I1) was able to convert crocetin dialdehyde into the crocin precursor crocetin. CsALDH3I1 carries a carboxyl-terminal hydrophobic domain, similar to that of the Neurospora crassa membrane-associated apocarotenoid dehydrogenase YLO-1. We also characterized the UDP-glycosyltransferase CsUGT74AD1, which converts crocetin to crocins 1 and 2'. In vitro assays revealed high specificity of CsALDH3I1 for crocetin dialdehyde and long-chain apocarotenals and of CsUGT74AD1 for crocetin. Following extract fractionation, CsCCD2, CsALDH3I1, and CsUGT74AD1 were found in the insoluble fraction, suggesting their association with membranes or large insoluble complexes. Analysis of protein localization in both C. sativus stigmas and following transgene expression in Nicotiana benthamiana leaves revealed that CsCCD2, CsALDH3I, and CsUGT74AD1 were localized to the plastids, the endoplasmic reticulum, and the cytoplasm, respectively, in association with cytoskeleton-like structures. Based on these findings and current literature, we propose that the endoplasmic reticulum and cytoplasm function as transit centers for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.

Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study.

BACKGROUND: Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS: We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. CONCLUSIONS: Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.