Nunzia Scotti

Genome-Wide Analysis of Plastid Gene Expression in Potato Leaf Chloroplasts and Tuber Amyloplasts: Transcriptional and Posttranscriptional Control

Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2- to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.

Virus-like particles produced in plants as potential vaccines

Virus-like particles (VLPs) have been produced as candidate vaccines in plants virtually since the introduction of biofarming. Even today, VLPs remain the best candidates for safe, immunogenic, efficacious and inexpensive vaccines. Well-characterized human animal viruses such as HBV, HCV, HIV and HPV, rotaviruses, norovirus, foot and mouth disease viruses and even influenza virus proteins have all been successfully investigated for VLP formation. Proteins have been produced in transgenic plants and via transient expression techniques; simple structures, structures depending on more than one protein, naked and enveloped particles have all been made. There have been multiple proofs of concept, more than a few proofs of efficacy, and several products moved into human trials. This review will cover the history of VLP production in plants, and will explore a few examples in detail to illustrate the potential of such a mode of production for human and animal medicine.

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.

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Δ9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Δ9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the “binding-type” vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

Organelle DNA analysis of Solanum and Brassica somatic hybrids by PCR with ’universal primers’

Abstract In order to set up a quick and easy procedure for determining the cytoplasmic composition of somatic hybrids, we tested a set of ’universal primers’ for plastidial and mitochondrial DNA on 13 genotypes belonging to the following species: Nicotiana tabacum, Solanum commersonii, Solanum tuberosum, Solanum etuberosum, Solanum phureja, Brassica oleracea, Brassica rapa, ’Anand’ CMS B. rapa, ’Chiang’ CMS B. oleracea, and ’Ogura’ CMS B. oleracea. Such primers are homologous to conserved coding sequences and amplify polymorphic intergenic or intronic regions. cpDNA polymorphism within Solanum and Brassica spp. was found with two and four primer pairs, respectively. The primers for the intergenic region between the trnF and trnV genes gave polymorphism among several tested species and were used in S. commersonii (+) S. tuberosum somatic hybrids,and B. oleracea (+) ’Anand’ CMS B. rapacybrids. Two primer pairs for mtDNA revealed polymorphism between S. commersonii and S. tuberosum, and one showed intraspecific polymorphism in S. tuberosum. The primer pair for the intergenic region between the rps14 and cob genes (pumD) showed a fragment of about 1.5 kb in S. tuberosum and S. phureja. A shorter fragment and no amplification were found in S. etuberosum and S. commersonii, respectively, suggesting frequent intrageneric rearrangements in this genome region. All Brassicaceae evidenced a fragment about 150-bp longer than in S. tuberosum. The same primers were also used with interspecific Solanum spp. somatic hybrids. Both PCR with pumD primers and hybridization with rpl5/rps14 genes indicated lack of linkage between rpl5/rps14 and cob genes in S. commersonii. Compared to direct visualization of restricted organellar DNA or Southern analysis with labelled probes, amplification of cpDNA and mtDNA with universal primers, followed by electrophoresis of either entire or restricted amplified fragments, is a simpler, more rapid and less expensive method to determine the organelle genome composition of interspecific Solanum and Brassica somatic hybrids.

Production of foreign proteins using plastid transformation.

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

Organelle DNA variation in parental Solanum spp. genotypes and nuclear-cytoplasmic interactions in Solanum tuberosum (+) S. commersonii somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC1 clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.

Improvement of plastid transformation efficiency in potato by using vectors with homologous flanking sequences.

Low transformation frequencies limit the use of plastid transformation in potato and other crops. Hence, a breakthrough in chloroplast genetic engineering of agronomically important species is a highly desirable goal. We succeeded in achieving potato transformation efficiency up to one shoot every bombardment using a modified regeneration procedure and 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. Such efficiency corresponds to 15-18-fold improvement compared to previous results obtained in potato with a progenitor vector of that used in our study, and is comparable to that usually achieved with tobacco. The results obtained represent a significant advancement towards the implementation of the plastid transformation technology in potato breeding and biotechnology.