Phytoene Desaturase Research Articles

Virus-induced gene silencing and virus-induced flowering in strawberry (Fragaria\u2009\xd7\u2009ananassa) using apple latent spherical virus vectors

Apple latent spherical virus (ALSV) vector is a convenient alternative to genetic transformation in horticultural plants, especially in species recalcitrant to genetic transformation. ALSV, an RNA virus, can infect a wide variety of plant species including major horticultural plants without inducing symptoms. Here, methodologies were developed for infection of ALSV vectors to strawberry seedlings and plantlets cultured in vitro. A seed-propagated F1 hybrid strawberry cultivar 'Yotsuboshi' was aseptically grown on half-strength Murashige–Skoog medium for 1 month and true leaves were inoculated with an ALSV RNA preparation by particle bombardment. ALSV vector infection rates varied from 58 to 100% according to the insertion sequences, in ‘Yotsuboshi’ seedlings. Plantlets (‘Dover’) propagated in vitro could also be infected with ALSV vector at a similar infection rate. For virus-induced gene silencing (VIGS), we prepared an ALSV vector carrying a 201 nucleotide segment of the strawberry phytoene desaturase gene. ‘Yotsuboshi’ and ‘Dover’ plants infected by this vector generated completely white leaves at fifth or sixth true leaves and above. For virus-induced flowering (VIF), we used an ALSV vector expressing the Arabidopsis thaliana flowering locus T gene. Strawberry seedlings infected by this vector started to flower from about 2 months post inoculation and bore fruits with viable seeds. The ALSV vector was no longer detected in any of the seedlings from early-flowered strawberries. Thus, the ALSV vector may be beneficial for examination of gene functions by VIGS in strawberry, and VIF using ALSV vector constitutes an effective new plant breeding technique for the promotion of cross-breeding in strawberry.

Implication of salt stress induces changes in pigment production, antioxidant enzyme activity, and qRT-PCR expression of genes involved in the biosynthetic pathway of Bixa orellana L.

The effect of salt stress on pigment synthesis and antioxidant enzyme activity as well as in the genes involved in the biosynthetic pathway of bixin was studied. The 14-day germinated seedlings of Bixa orellana were induced into the various NaCl concentration (0, 25, 50, 75, 100mM). After 45days, leaves were taken for pigment analysis, antioxidant assays, and gene expression analysis to study the response of salt stress. The pigment content such as chlorophyll level was increased upon salt stress with a reduction in total carotenoid clearly indicating the adaptability of plants towards the stressed state. The level of β-carotene was increased in the highest concentration of salt stress treatment. The secondary metabolites such as bixin and abscisic acid (ABA) content were also high in elevated concentration of salt-treated seedling than control. The antioxidant enzyme activity was increased with the highest dose of salt stress suggesting the antioxidant enzymes to protect the plant from the deleterious effects. The mRNA transcript gene of the carotenoid biosynthetic pathway such as phytoene synthase (PSY), 1-deoxyxylulose-5-phosphate synthase (DXS), phytoene desaturase (PDS), beta-lycopene cyclase (LCY-β), epsilon lycopene cyclase (LCY-ε), carboxyl methyl transferase (CMT), aldehyde dehydrogenase (ADH), lycopene cleavage dioxygenase (LCD), and carotenoid cleavage dioxygenase (CCD) showed differential expression pattern under salt stress. In addendum, we studied the co-expression network analysis of gene to assess the co-related genes associated in the biosynthesis pathway of carotenoid. From the co-expression analysis result showed, the LCY, PDS, and PSY genes were closely correlated with other genes. These finding may provide insight to the plants to exist in the stress condition and to improve the industrially important pigment production.

Construction of a Nonnatural C60 Carotenoid Biosynthetic Pathway.

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C60 carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient synthases for C60 carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate synthase, we observed that C60-phytoene accumulated together with a small amount of C65-phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids failed to serve as a substrate for carotene desaturases, the C25-half of the C55-phytoene was accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids, which are promising components for various biological (light-harvesting, antioxidant, and communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.

Virus-induced gene silencing in Nicotiana benthamiana triggered by heterologous gene sequences from Viola philippica

Virus-induced gene silencing (VIGS) is a particularly useful tool for functional genomics. In the present study, we attempted to utilize this technology to infer the function of genes from Viola philippica using a tobacco rattle virus (TRV) construct. Firstly, the phytoene desaturase gene from V. philippica (VpPDS) was silenced, and local leaf bleaching was observed but did not exhibit systemic effects, thereby limiting utilization of TRV-mediated gene silencing in the recipient plant. However, we observed systemic gene silencing in Nicotiana benthamiana when the VpPDS sequence was used as a trigger, thereby suggesting that heterologous gene sequences could elicit gene silencing. We then investigated the role of gene PISTILLATA from V. philippica (VpPI) of the B-class MADS-box gene family, which regulates the identity and development of stamens and petals. Using the coding region of VpPI as triggers, we determined the gene silencing efficiency of the corresponding GLOBOSA paralogs in N. benthamiana (NbGLO1 and NbGLO2), and we observed stamen-to-carpel transformation and distorted corollas in N. benthamiana suggesting that VpPI functioned as the NbGLO gene. However, the 3'-untranslated region (3'UTR) of VpPI and each 3'UTR of the NbGLO genes downregulated its own corresponding gene, hardly producing floral homeotic alterations. This work provides a better understanding of gene-specific probe design for gene silencing as well as shows that heterologous sequence-triggered VIGS is an efficient way to investigate functional conservation of orthologous genes.

No apparent fitness costs associated with phytoene desaturase mutations conferred resistance to diflufenican and picolinafen in oriental mustard (Sisymbrium orientale L.)

Two mutations Leu498 and Glu425 in the PDS gene were identified as the main cause conferring resistance to diflufenican and picolinafen in two oriental mustard populations P3 and P40. As mutations are suspected to affect fitness, this study was designed to test this hypothesis using the F2 of two crosses P3.2 (P3♂ × S♀) and P40.5 (P40♂ × S♀) of oriental mustard. The F2 plants, which segregated for target-site point mutations of PDS gene (Leu498 and Glu425) grown in monoculture and under competition with wheat in pot-trials and evaluated for growth and fecundity. All F2 individuals were genotyped by using Cleaved Amplified Polymorphic Sequence (CAPS) technique. Regression analysis showed no fitness cost in the resistant plants because no significant difference was identified in seed and biomass production within RR, RS and SS individuals. The absence of measurable negative effects on fitness associated mutations suggests that the frequency of the PDS resistance alleles will not decline in the absence of selection pressure of PDS-inhibitors.

Exploring the differential mechanisms of carotenoid biosynthesis in the yellow peel and red flesh of papaya

BackgroundRed-fleshed papaya is a good material to study the different carotenoids accumulation mechanism in the peel and flesh. Although the peel and flesh of papaya closely integrated into one body, the flesh coloration changing from white to red, while the exocarp coloration changing from green to yellow. In this study, the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes in the process of papaya fruit ripening were studied, and the carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were investigated.ResultsThe carotenoid composition in papaya flesh and peel were different. The major carotenoids were lutein and β-carotene in the peel, while lycopene in the flesh. The accumulation of carotenoids, including lycopene, β-carotene, and β-cryptoxanthin were considered to cause the orange-red color of papaya cv. ‘Daqing No.10’ flesh. The color of peel changed from green to yellow because of the fast degradation of chlorophyll and the appearance of carotenoids such as lutein and β-carotene. Thirteen genes that encode enzymes in the carotenoid biosynthetic pathway were detected in papaya fruit transcriptome: two phytoene synthase (PSY1, PSY2), two phytoene desaturase (PDS1, PDS2), one ζ-carotene desaturase (ZDS), four lycopene cyclase (CYCB, LCYB1, LCYB2, LCYE), one β-carotene hydroxylase (CHYB), one carotene ε-monooxygenase (LUT1), one violaxanthin de-epoxidase (VDE), and one zeaxanthin epoxidase (ZEP). The results of RNA-Seq and RT-qPCR showed the expression of carotenoid biosynthetic pathway genes was consistent with the change of carotenoid content. Carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were analysed based on the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes. There was only a β-branch of carotenoid biosynthesis in the flesh of papaya, while there were both α- and β-branch of carotenoid biosynthesis in papaya peel. In the process of papaya fruit ripening, the α-branch was inhibited and the β-branch was enhanced in the peel.ConclusionsThe differential carotenoid accumulation and biosynthesis pathway genes expression in peel and flesh, lay a foundation for further study and provide further insights to control fruit color and improve fruit quality and appearance.

Differential transcriptional responses of carotenoid biosynthesis genes in the marine green alga Tetraselmis suecica exposed to redox and non-redox active metals.

The green microalga, Tetraselmis suecica, is commonly used in scientific, industrial, and aquacultural purposes because of its high stress tolerance and ease of culture in wide spectrums of environments. We hypothesized that carotenoids help to protect Tetraselmis cells from environmental stress by regulating genes in biosynthetic pathways. Here, we determined three major carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), and β-lycopene cyclase (LCY-B) in T. suecica, and examined the physiological parameters and gene expression responses when exposed to redox-active metals and non-redox-active metals. Phylogenetic analyses of each gene indicated that T. suecica clustered well with other green algae. Real-time PCR analysis showed that TsPSY, TsPDS, and TsLCY-B genes greatly responded to the redox-active metals in CuSO4 followed by CuCl2, but not to the non-redox-active metals. The redox-active metals strongly affected the physiology of the cells, as determined by cell counting, reactive oxygen species (ROS) imaging, and photosynthetic efficiency. This suggests that carotenoids protect the cells from oxidative damage caused by metals, thereby contributing to cell survival under various stress conditions.

Genome Editing by CRISPR/Cas9 in Sorghum Through Biolistic Bombardment.

The advanced biotechnology CRISPR/Cas9 as a powerful genome editing tool has displayed great potential for improving important agronomic traits such as yield and quality. It has gained momentum worldwide for gene function research of plants in recent years. As for cereals, numerous studies of CRISPR/Cas9 have been reported predominately on rice and quite a few on other cereals including maize, wheat, and barley. In contrast, there are only a couple of reports on sorghum up to date. In this chapter, the CRISPR/Cas9 system has been investigated for sorghum genome editing through biolistic bombardment. Two target genes, cinnamyl alcohol dehydrogenase (CAD) and phytoene desaturase (PDS), have been investigated by CRISPR/Cas9 though bomboarment. Successful genome editing has been achieved within the sorghum genotype Tx430. Furthermore, sequencing PCR product of transgenic plants has confirmed that the CRISPR/Cas9 successfully edited the target gene in sorghum. Both homozygosis and heterozygosis editings of CAD gene have been confirmed in T0 primary transgenic lines through sequencing PCR products. T1 generation of CRISPR plants has been investigated as well. The results illustrated that the edited gene has passed down to next generation. More experiments, such as optimizing promoters for guide RNA (gRNA) and Cas9 in sorghum, are under investigation. Three factors were considered crucial elements to establish an efficient CRISPR/Cas9 system for genome editing in sorghum: (1) an efficient transformation system, (2) the design of targeted gene sequence for gRNA, (3) effective expression of CRISPR components including Cas9 and gRNA.

Engineering Haloferax mediterranei as an Efficient Platform for High Level Production of Lycopene.

Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax mediterranei, a halophilic archaeon, as a new lycopene producer. H. mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature.

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

Metabolic Engineering of Deinococcus radiodurans for the Production of Phytoene.

A metabolically-engineered Deinococcus radiodurans R1 strain capable of producing phytoene, a colorless C40 carotenoid and a promising antioxidant, has been developed. To make this base strain, first, the crtI gene encoding phytoene desaturase was deleted to block the conversion of phytoene to other carotenoids such as lycopene and γ-carotene. This engineered strain produced 0.413 ± 0.023 mg/l of phytoene from 10 g/l of fructose. Further enhanced production of phytoene up to 4.46 ± 0.19 mg/l was achieved by overexpressing the crtB gene encoding phytoene synthase and the dxs genes encoding 1-deoxy-D-xylulose-5-phosphate synthase gene, and by deleting the crtD gene. High cell-density culture of our final engineered strain allowed production of 10.3 ± 0.85 mg/l of phytoene with the yield and productivity of 1.04 ± 0.05 mg/g and 0.143 ± 0.012 mg/l/h, respectively, from 10 g/l of fructose. Furthermore, the antioxidant potential of phytoene produced by the final engineered strain was confirmed by in vitro DPPH radical-scavenging assay.

High hydrostatic pressure treatments trigger de novo carotenoid biosynthesis in papaya fruit (Carica papaya cv. Maradol).

High hydrostatic pressure (HHP) processing is a non-thermal technology reported to increase desirable metabolites in plant foods. This work evaluated changes in carotenoid accumulation in fresh-cut papaya fruit as affected by HHP treatment (50-400 MPa for 3-60 min) and during subsequent storage at 4 °C; simultaneously, transcriptional activities of carotenoid biosynthetic genes and oxidative stress markers were evaluated. LC-MS analyses revealed that HHP treatment increased carotenoid precursors and carotenes contents following processing and storage: lycopene levels increased up to 11-fold compared to the non-treated samples, and H2O2 and lipid peroxidation were concomitantly increased. qRT-PCR of intact RNA showed that the amount of phytoene desaturase transcripts increased after HHP treatment, and that they were correlated with carotene accumulation. This is the first study to show that HHP treatment triggers de novo carotenoid biosynthesis, which is regulated at the transcriptional level, possibly by inducing oxidative stress signaling in fruit tissue.

Low Temperature Stress Alters the Expression of Phytoene Desaturase Genes (crtP1 and crtP2) and the ζ-Carotene Desaturase Gene (crtQ) Together with the Cellular Carotenoid Content of Euglena gracilis.

Carotenoids participate in photosynthesis and photoprotection in oxygenic phototrophs. Euglena gracilis, a eukaryotic phytoflagellate, synthesizes several carotenoids: β-carotene, neoxanthin, diadinoxanthin and diatoxanthin. Temperature is one of the most striking external stimuli altering carotenoid production. In the present study, to elucidate the regulation of carotenoid synthesis of E. gracilis in response to environmental stimuli, we functionally identified phytoene desaturase genes (crtP1 and crtP2) and the ζ-carotene desaturase gene (crtQ) of this alga and analyzed expression of those genes and the composition of major carotenoids in cells grown under cold (20�C) and high-intensity light (HL; 240 �mol photon m-2 s-1) conditions. 20�C-HL treatment increased the transcriptional level of the phytoene synthase gene (crtB), and crtP1 and crtP2, whose products catalyze the early steps of carotenoid biosynthesis in this alga. Cultivation at 20�C under illumination at 55 �mol photon m-2 s-1 (low-intensity light; LL) decreased the cell concentration, Chl and total major carotenoid content by 61, 75 and 50%, respectively, relative to control (25�C-LL) cells. When grown at 20�C-HL, the cells showed a greater decrease in cell concentration and photosynthetic pigment contents than those in 20�C-LL. β-Carotene, neoxanthin and diadinoxanthin contents were decreased by more than half in 20�C-LL and 20�C-HL treatments. On the other hand, when subjected to 20�C-LL and 20�C-HL, the cells retained a diatoxanthin content comparable with control cells. Our findings suggested that diatoxanthin plays crucial roles in the acclimation to cold and intense light condition. To the best of our knowledge, this is the first report on a photosynthetic organism possessing dual crtP genes.

Virus-induced Phytoene Desaturase (PDS) Gene Silencing Using Tobacco Rattle Virus in Lilium × formolongi

Gene function analysis is challenging for Lilium due to the lack of an efficient method for stable genetic transformation. Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. This study reported that Tobacco rattle virus (TRV)-based VIGS of a PHYTOENE DESATURASE (PDS) ortholog (LhPDS) can be achieved in Lilium × formolongi seedlings, with a survival rate of 92%, using the inoculation method of rubbing plus injection. Compared with untreated and mock-treated seedlings, the photobleached leaf phenotype of silenced plants significantly correlates with down-regulation of endogenous LhPDS (P ≤ 0.05). In addition, the silencing phenomenon can be observed in the growing points of plants, indicating that systemic viral infection was achieved using the protocol. The results indicate that TRV-based VIGS can be used to characterize gene function in Lilium × formolongi. This work lays the foundation for gene function analysis and molecular breeding in Lilium spp.

From Golden Rice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, β-carotene ketolase, and β-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained β-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.

Agroinfiltration of Phytoene Desaturase and Lycopene Β-Cyclase Genes from Bacterial Source in Tomato (Solanum Lycopersicum L.) Enhances Nutritional and Processing Quality of its Juice

ABSTRACTThere has been considerable interest in the value of carotenoids due to their high nutritional and nutraceutical properties. We have earlier shown that agroinfiltration with Agrobacterium tumifaciens GV 3101 containing binary vector pRI 101(I) harboring CrtI gene (Phytoene desaturase) and binary vector pRI 101(Y) harboring CrtY gene (Lycopene β-cyclase) from bacterial sources increases carotenoids in tomatoes. In the present study, the juice prepared from fruits having CrtY gene had higher content of β-carotene (0.60 mg 100 mL−1), lycopene (0.40 mg 100 mL−1) and total phenolics (11.41 μg GAE mL−1) compared to juice prepared from control fruits (0.47 mg 100 mL−1, 0.32 mg 100 mL−1 and 11.22 μg GAE mL−1, respectively), and it showed better antioxidant potential (DPPH IC50 value of 27.6) than that of non-transgenic fruit juice (56.4 μL mL−1). The juice from fruits having CrtY gene also had lower Bostwick value (7.5 cm min−1) than control fruits (9.8 cm min−1), indicating improvement in processing quality because of gene integration. Juice prepared from agroinfiltrated tomatoes may benefit both consumers as well as juice processing industry due to their high nutritional value and better processing quality.

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method.

A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477-7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629-8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5'end without changing the amino acid sequence. The syn-pds-int gene (1872bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy.

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9-based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site-specific mutations in Tragopogon protoplasts. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9-based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

An optimized method and a dominant selectable marker for genetic engineering of an industrially promising microalga—Pavlova lutheri

The marine microalga Pavlova lutheri has great nutritional value as it synthesizes and accumulates higher amounts of polyunsaturated fatty acids (PUFAs). It is commonly used in aquaculture. However, no transformation procedure has been realized for this industrially important microalga so far. Here, we report an efficient protocol for the nuclear transformation of P. lutheri. Agrobacterium-mediated transformation (AMT) of P. lutheri with a mutated genomic clone of phytoene desaturase (pds) gene, pds-L504R, from Haematococcus pluvialis yielded norflurazon-resistant P. lutheri cells. Ideal co-cultivation conditions for achieving higher numbers of transformants was found to be artificial seawater (ASW) medium, 100 μM acetosyringone, and a 24-h co-cultivation at 25 ± 1 °C. The integration of the introduced gene into the nuclear genome of P. lutheri was shown by PCR amplification of the T-DNA sequences from the genomic DNA of transformants and Southern blot analysis using T-DNA sequences as probes. The transgene expressed efficiently as evidenced by the results of stability and tolerance study, and the qRT-PCR analysis. Results clearly demonstrate the application of AMT approach and pds gene as a dominant selectable marker for the genetic engineering of P. lutheri for fundamental studies and biotechnological applications.

Disruption of Chloroplast Function Through Downregulation of Phytoene Desaturase Enhances the Systemic Accumulation of an Aphid-Borne, Phloem-Restricted Virus.

Chloroplasts play a central role in pathogen defense in plants. However, most studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae includes RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins that is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll, and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.