Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

KEY MESSAGE: We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.

Genome wide association mapping for agronomic, fruit quality, and root architectural traits in tomato under organic farming conditions.

BACKGROUND: Opportunity and challenges of the agriculture scenario of the next decades will face increasing demand for secure food through approaches able to minimize the input to cultivations. Large panels of tomato varieties represent a valuable resource of traits of interest under sustainable cultivation systems and for genome-wide association studies (GWAS). For mapping loci controlling the variation of agronomic, fruit quality, and root architecture traits, we used a heterogeneous set of 244 traditional and improved tomato accessions grown under organic field trials. Here we report comprehensive phenotyping and GWAS using over 37,300 SNPs obtained through double digest restriction-site associated DNA (dd-RADseq). RESULTS: A wide range of phenotypic diversity was observed in the studied collection, with highly significant differences encountered for most traits. A variable level of heritability was observed with values up to 69% for morphological traits while, among agronomic ones, fruit weight showed values above 80%. Genotype by environment analysis highlighted the strongest genotypic effect for aboveground traits compared to root architecture, suggesting that the hypogeal part of tomato plants has been a minor objective for breeding activities. GWAS was performed by a compressed mixed linear model leading to 59 significantly associated loci, allowing the identification of novel genes related to flower and fruit characteristics. Most genomic associations fell into the region surrounding SUN, OVATE, and MYB gene families. Six flower and fruit traits were associated with a single member of the SUN family (SLSUN31) on chromosome 11, in a region involved in the increase of fruit weight, locules number, and fruit fasciation. Furthermore, additional candidate genes for soluble solids content, fruit colour and shape were found near previously reported chromosomal regions, indicating the presence of synergic and multiple linked genes underlying the variation of these traits. CONCLUSIONS: Results of this study give new hints on the genetic basis of traits in underexplored germplasm grown under organic conditions, providing a framework for the development of markers linked to candidate genes of interest to be used in genomics-assisted breeding in tomato, in particular under low-input and organic cultivation conditions.

Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

Progress of targeted genome modification approaches in higher plants.

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.

An association mapping approach to identify favourable alleles for tomato fruit quality breeding.

BACKGROUND: Genome Wide Association Studies (GWAS) have been recently used to dissect complex quantitative traits and identify candidate genes affecting phenotype variation of polygenic traits. In order to map loci controlling variation in tomato marketable and nutritional fruit traits, we used a collection of 96 cultivated genotypes, including Italian, Latin American, and other worldwide-spread landraces and varieties. Phenotyping was carried out by measuring ten quality traits and metabolites in red ripe fruits. In parallel, genotyping was carried out by using the Illumina Infinium SolCAP array, which allows data to be collected from 7,720 single nucleotide polymorphism (SNP) markers. RESULTS: The Mixed Linear Model used to detect associations between markers and traits allowed population structure and relatedness to be evidenced within our collection, which have been taken into consideration for association analysis. GWAS identified 20 SNPs that were significantly associated with seven out of ten traits considered. In particular, our analysis revealed two markers associated with phenolic compounds, three with ascorbic acid, ß-carotene and trans-lycopene, six with titratable acidity, and only one with pH and fresh weight. Co-localization of a group of associated loci with candidate genes/QTLs previously reported in other studies validated the approach. Moreover, 19 putative genes in linkage disequilibrium with markers were found. These genes might be involved in the biosynthetic pathways of the traits analyzed or might be implied in their transcriptional regulation. Finally, favourable allelic combinations between associated loci were identified that could be pyramided to obtain new improved genotypes. CONCLUSIONS: Our results led to the identification of promising candidate loci controlling fruit quality that, in the future, might be transferred into tomato genotypes by Marker Assisted Selection or genetic engineering, and highlighted that intraspecific variability might be still exploited for enhancing tomato fruit quality.

Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events.

BACKGROUND: The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). RESULTS: Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. CONCLUSIONS: Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Whole-genome resequencing reveals genomic footprints of Italian sweet and hot pepper heirlooms giving insight into genes underlying key agronomic and qualitative traits.

BACKGROUND: Pepper is a major crop species of the Solanaceae family, largely appreciated for its high nutritional and healthy contribution to human diets. In the Mediterranean basin, the favorable pedoclimatic conditions enhanced the selection of several diversified landraces cultivated pepper (Capsicum annuum), for whom Italy can be considered a main pole of diversification. Hence, a survey of traditional C. annuum genetic resources is essential for deep understanding of such diversity and for applications in genomics assisted breeding. Here, we report whole-genome resequencing analyses of two sweet and two pungent genotypes highly diffused in South Italy and representative of the variability for shape, colour and nutritional properties. RESULTS: The four genomes were reconstructed at a chromosomal scale using a reference-guided approach, based on a dataset of 2.6 billion paired-end reads, corresponding to 20× genome coverage and a mapping rate above 99% for a final genomes size of approximately 3 Gb. After five iterations of variant calling, a total of 29,258,818 single nucleotide polymorphisms (SNPs) and 1,879,112 InDels, were identified. Substantial differences were observed among the four genomes based on geographical origin, with chromosomes 9 and 11 showing more polymorphisms in the accessions with higher fruit weight and absence of pungency. Among the identified variants, a small private indel (T - > TA) shared between sweet and big fruits accessions induces a frameshift with the generation of a new stop codon in a gene annotated as extensin, whereas two private SNPs within hot types were identified in 1-aminocyclopropane-1-carboxylate oxidase (ACO), a key gene involved in fruit ripening. The estimation of repetitive elements highlights a preponderant presence of Long Terminal Repeats (LTRs), the majority of which belonged to Gypsy superfamily. By comparing the four genomes with publicly available references including 'CM334' and Zunla-1 highlight the presence of 49,475 shared gene families. CONCLUSIONS: The new genomic sequences aim to enrich the whole genome information of pepper local varieties, providing a valuable tool for precision gene mapping, marker discovery, comparative studies. Such knowledge widens the frontiers to understand the selection history of Italian pepper landraces toward the recognition of specificity local agri-food products marks.

Surveying soil-borne disease development on wild rocket salad crop by proximal sensing based on high-resolution hyperspectral features.

Wild rocket (Diplotaxis tenuifolia, Brassicaceae) is a baby-leaf vegetable crop of high economic interest, used in ready-to-eat minimally processed salads, with an appreciated taste and nutraceutical features. Disease management is key to achieving the sustainability of the entire production chain in intensive systems, where synthetic fungicides are limited or not permitted. In this context, soil-borne pathologies, much feared by growers, are becoming a real emergency. Digital screening of green beds can be implemented in order to optimize the use of sustainable means. The current study used a high-resolution hyperspectral array (spectroscopy at 350-2500 nm) to attempt to follow the progression of symptoms of Rhizoctonia, Sclerotinia, and Sclerotium disease across four different severity levels. A Random Forest machine learning model reduced dimensions of the training big dataset allowing to compute de novo vegetation indices specifically informative about canopy decay caused by all basal pathogenic attacks. Their transferability was also tested on the canopy dataset, which was useful for assessing the health status of wild rocket plants. Indeed, the progression of symptoms associated with soil-borne pathogens is closely related to the reduction of leaf absorbance of the canopy in certain ranges of visible and shortwave infrared spectral regions sensitive to reduction of chlorophyll and other pigments as well as to modifications of water content and turgor.

Sorting biotic and abiotic stresses on wild rocket by leaf-image hyperspectral data mining with an artificial intelligence model.

BACKGROUND: Wild rocket (Diplotaxis tenuifolia) is prone to soil-borne stresses under intensive cultivation systems devoted to ready-to-eat salad chain, increasing needs for external inputs. Early detection of the abiotic and biotic stresses by using digital reflectance-based probes may allow optimization and enhance performances of the mitigation strategies. METHODS: Hyperspectral image analysis was applied to D. tenuifolia potted plants subjected, in a greenhouse experiment, to five treatments for one week: a control treatment watered to 100% water holding capacity, two biotic stresses: Fusarium wilting and Rhizoctonia rotting, and two abiotic stresses: water deficit and salinity. Leaf hyperspectral fingerprints were submitted to an artificial intelligence pipeline for training and validating image-based classification models able to work in the stress range. Spectral investigation was corroborated by pertaining physiological parameters. RESULTS: Water status was mainly affected by water deficit treatment, followed by fungal diseases, while salinity did not change water relations of wild rocket plants compared to control treatment. Biotic stresses triggered discoloration in plants just in a week after application of the treatments, as evidenced by the colour space coordinates and pigment contents values. Some vegetation indices, calculated on the bases of the reflectance data, targeted on plant vitality and chlorophyll content, healthiness, and carotenoid content, agreed with the patterns of variations observed for the physiological parameters. Artificial neural network helped selection of VIS (492-504, 540-568 and 712-720 nm) and NIR (855, 900-908 and 970 nm) bands, whose read reflectance contributed to discriminate stresses by imaging. CONCLUSIONS: This study provided significative spectral information linked to the assessed stresses, allowing the identification of narrowed spectral regions and single wavelengths due to changes in photosynthetically active pigments and in water status revealing the etiological cause.

Genotypic and Environmental Effects on Morpho-Physiological and Agronomic Performances of a Tomato Diversity Panel in Relation to Nitrogen and Water Stress Under Organic Farming.

The agricultural scenario of the upcoming decades will face major challenges for the increased and sustainable agricultural production and the optimization of the efficiency of water and fertilizer inputs. Considering the current and foreseen water scarcity in several marginal and arid areas and the need for a more sustainable farming production, the selection and development of cultivars suitable to grow under low-input conditions is an urgent need. In this study, we assayed 42 tomato genotypes for thirty-two morpho-physiological and agronomic traits related to plant, fruit, and root characteristics under standard (control) and no-nitrogen fertilization or water deficit (30% of the amount given to non-stressed trials) treatments in two sites (environments), which corresponded to organic farms located in Italy and Spain. A broad range of variation was found for all traits, with significant differences between the applied treatments and the cultivation sites. Dissection of genotypic (G), environmental (E), and treatment (T) factors revealed that the three main factors were highly significant for many traits, although G was the main source of variation in most cases. G × E interactions were also important, while G × T and E × T were less relevant. Only fruit weight and blossom end rot were highly significant for the triple interaction (G × E × T). Reduction of water supply significantly increased the soluble solid content in both locations, whereas both nitrogen and water stress led to a general decrease in fruit weight and total yield. Despite so, several accessions exhibited better performances than the control when cultivated under stress. Among the accessions evaluated, hybrids were promising in terms of yield performance, while overall landraces and heirlooms exhibited a better quality. This suggests the possibility of exploiting both the variation within ancient varieties and the heterosis for yield of hybrids to select and breed new varieties with better adaptation to organic farming conditions, both under optimal and suboptimal conditions. The results shed light on the strategies to develop novel varieties for organic farming, giving hints into the management of inputs to adopt for a more sustainable tomato cultivation.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5' and 3' regulatory sequences.

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5'-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5'-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5'-UTR construct, described above, and another containing the same terminator, but with the promoter and 5'-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Plastid Transformation in Potato: An Important Source of Nutrition and Industrial Materials.

For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.

Functional Hyperspectral Imaging by High-Related Vegetation Indices to Track the Wide-Spectrum Trichoderma Biocontrol Activity Against Soil-Borne Diseases of Baby-Leaf Vegetables.

Research has been increasingly focusing on the selection of novel and effective biological control agents (BCAs) against soil-borne plant pathogens. The large-scale application of BCAs requires fast and robust screening methods for the evaluation of the efficacy of high numbers of candidates. In this context, the digital technologies can be applied not only for early disease detection but also for rapid performance analyses of BCAs. The present study investigates the ability of different Trichoderma spp. to contain the development of main baby-leaf vegetable pathogens and applies functional plant imaging to select the best performing antagonists against multiple pathosystems. Specifically, sixteen different Trichoderma spp. strains were characterized both in vivo and in vitro for their ability to contain R. solani, S. sclerotiorum and S. rolfsii development. All Trichoderma spp. showed, in vitro significant radial growth inhibition of the target phytopathogens. Furthermore, biocontrol trials were performed on wild rocket, green and red baby lettuces infected, respectively, with R. solani, S. sclerotiorum and S. rolfsii. The plant status was monitored by using hyperspectral imaging. Two strains, Tl35 and Ta56, belonging to T. longibrachiatum and T. atroviride species, significantly reduced disease incidence and severity (DI and DSI) in the three pathosystems. Vegetation indices, calculated on the hyperspectral data extracted from the images of plant-Trichoderma-pathogen interaction, proved to be suitable to refer about the plant health status. Four of them (OSAVI, SAVI, TSAVI and TVI) were found informative for all the pathosystems analyzed, resulting closely correlated to DSI according to significant changes in the spectral signatures among health, infected and bio-protected plants. Findings clearly indicate the possibility to promote sustainable disease management of crops by applying digital plant imaging as large-scale screening method of BCAs' effectiveness and precision biological control support.

ddRAD sequencing-based genotyping for population structure analysis in cultivated tomato provides new insights into the genomic diversity of Mediterranean 'da serbo' type long shelf-life germplasm.

Double digest restriction-site associated sequencing (ddRAD-seq) is a flexible and cost-effective strategy for providing in-depth insights into the genetic architecture of germplasm collections. Using this methodology, we investigated the genomic diversity of a panel of 288 diverse tomato (Solanum lycopersicum L.) accessions enriched in 'da serbo' (called 'de penjar' in Spain) long shelf life (LSL) materials (152 accessions) mostly originating from Italy and Spain. The rest of the materials originate from different countries and include landraces for fresh consumption, elite cultivars, heirlooms, and breeding lines. Apart from their LSL trait, 'da serbo' landraces are of remarkable interest for their resilience. We identified 32,799 high-quality SNPs, which were used for model ancestry population structure and non-parametric hierarchical clustering. Six genetic subgroups were revealed, clearly separating most 'da serbo' landraces, but also the Spanish germplasm, suggesting a subdivision of the population based on type and geographical provenance. Linkage disequilibrium (LD) in the collection decayed very rapidly within <5 kb. We then investigated SNPs showing contrasted minor frequency allele (MAF) in 'da serbo' materials, resulting in the identification of high frequencies in this germplasm of several mutations in genes related to stress tolerance and fruit maturation such as CTR1 and JAR1. Finally, a mini-core collection of 58 accessions encompassing most of the diversity was selected for further exploitation of key traits. Our findings suggest the presence of a genetic footprint of the 'da serbo' germplasm selected in the Mediterranean basin. Moreover, we provide novel insights on LSL 'da serbo' germplasm as a promising source of alleles for tolerance to stresses.

Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes.

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication.

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts.

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.