Cisgenic apple trees; development, characterization, and performance
Krens, F.A. ; Schaart, J.G. ; Burgh, A.M. van der; Tinnenbroek-Capel, I.E.M. ; Groenwold, R. ; Kodde, L.P. ; Broggini, G.A.L. ; Gessler, C. ; Schouten, H.J. - \ 2015
Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 11 p.
scab resistance gene - selectable marker - mediated transformation - plant transformation - transcription factor - transgenic apple - agrobacterium - gala - recombinase - expression
Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.
Molecular cloning and characterization of the trichome specificchrysanthemyl diphosphate/chrysanthemol synthase promoter fromTanacetum cinerariifolium
Sultana, S. ; Hu, H. ; Gao, L. ; Mao, J. ; Luo, J. ; Jongsma, M.A. ; Wang, C. - \ 2015
Scientia Horticulturae 185 (2015). - ISSN 0304-4238 - p. 193 - 199.
mosaic virus-35s promoter - artemisia-annua - transcription factor - diphosphate synthase - pyrethrin biosynthesis - transgene expression - plant transformation - glandular trichomes - gene - cinerariaefolium
Natural pyrethrins accumulate to high concentrations in the flower achenes of pyrethrum (Tanacetumcinerariifolium). They are extracted from dried flowers and widely used as a natural pesticide. Chrysanthe-mol synthase (CHS) is the first key enzyme in the biosynthesis of pyrethrins. In this work, a 1128 bp TcCHSpromoter fragment was cloned from pyrethrum genomic DNA. The sequence contained cis-elementspredicted to be responsive to different hormones, light, and environmental stresses. To characterizethe promoter it was fused to the reporter genes green fluorescent protein (GFP) and -glucuronidase(GUS), and respectively transformed into florist’s chrysanthemum (Chrysanthemum morifolium ‘1581’)and tobacco (Nicotiana tabacum). GFP fluorescence in florist’s chrysanthemum ‘1581’and GUS staining oftobacco showed that the TcCHS promoter was exclusively expressed in the glandular secretory trichomes(GSTs) of both plant species. The findings will support research on factors influencing the accumulationof pyrethrins, and can be used for trichome-specific metabolic engineering of plants to ensure minimaladverse effects on plant growth and development.
AIL and HDG proteins act antagonistically to control cell proliferation
Horstman, A. ; Fukuoka, H. ; Muino Acuna, J.M. ; Nitsch, L.M.C. ; Guo, Changhao ; Passarinho, P.A. ; Sanchez Perez, G.F. ; Immink, R.G.H. ; Angenent, G.C. ; Boutilier, K.A. - \ 2015
Development 142 (2015). - ISSN 0950-1991 - p. 454 - 464.
arabidopsis-thaliana - transcription factors - plant transformation - ectopic expression - quantitative pcr - chip-seq - differentiation - genes - plethora - growth
AINTEGUMENTA-LIKE (AIL) transcription factors are key regulators of cell proliferation and meristem identity. Although AIL functions have been well described, the direct signalling components of this pathway are largely unknown.We show that BABY BOOM(BBM) and other AIL proteins physically interact with multiple members of the L1-expressed HOMEODOMAIN GLABROUS (HDG) transcription factor family, including HDG1, HDG11 and HDG12. Overexpression of HDG1, HDG11 and HDG12 restricts growth due to root and shoot meristem arrest, which is associated with reduced expression of genes involved in meristem development and cell proliferation pathways, whereas downregulation of multiple HDG genes promotes cell overproliferation. These results suggest a role for HDG proteins in promoting cell differentiation. We also reveal a transcriptional network in which BBM andHDG1regulate several common target genes, and whereBBM/AIL and HDG regulate the expression of each other. Taken together, these results suggest opposite roles for AIL and HDG proteins, with AILs promoting cell proliferation and HDGs stimulating cell differentiation, and that these functions are mediated at both the protein-protein interaction and transcriptional level.
(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways
Cankar, K. ; Jongedijk, E.J. ; Klompmaker, M. ; Majdic, T. ; Mumm, R. ; Bouwmeester, H.J. ; Bosch, H.J. ; Beekwilder, M.J. - \ 2015
Biotechnology Journal 10 (2015)1. - ISSN 1860-6768 - p. 180 - 189.
plant transformation - biosynthetic-pathway - terpenoid metabolism - squalene synthase - tobacco - expression - artemisinin - arabidopsis - reductase - precursors
Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants. Keywords: 5-Epi-aristolochene synthase · Metabolic engineering · RNAi · Squalene synthase
An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato
Gomez Roldan, M.V. ; Outchkourov, N.S. ; Houwelingen, A.M.M.L. van; Lammers, M. ; Romero Fuente, I. ; Ziklo, N. ; Aharoni, A. ; Hall, R.D. ; Beekwilder, M.J. - \ 2014
The Plant Journal 80 (2014)4. - ISSN 0960-7412 - p. 695 - 708.
plant transformation - transcription factor - petunia-hybrida - fruit - biosynthesis - expression - protein - system - metabolome - infection
Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.
Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization
Dong, L. ; Miettinen, K. ; Verstappen, F.W.A. ; Voster, A. ; Jongsma, M.A. ; Memelink, J. ; Krol, S. van der; Bouwmeester, H.J. - \ 2013
Metabolic Engineering 20 (2013). - ISSN 1096-7176 - p. 198 - 211.
indole alkaloid pathway - gland secretory-cells - roseus hairy roots - catharanthus-roseus - monoterpene biosynthesis - isoprenoid biosynthesis - plant transformation - essential oils - grape juice - metabolism
Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.
Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight
Zhu, S. ; Duwal, A. ; Su, Q. ; Vossen, J.H. ; Visser, R.G.F. ; Jacobsen, E. - \ 2013
Transgenic Research 22 (2013)2. - ISSN 0962-8819 - p. 315 - 325.
agrobacterium-mediated transformation - phytophthora-infestans - backbone sequences - transgene expression - plant transformation - dna transfer - genome - rice - tumefaciens - chromosome
Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight
One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE
Untergasser, A. ; Bijl, G.J.M. ; Liu, W. ; Bisseling, T. ; Schaart, J.G. ; Geurts, R. - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203
site-specific recombination - root-nodule organogenesis - selectable marker gene - genomic dna fragments - medicago-truncatula - plant transformation - nodulation factors - lotus-japonicus - host-range - cytokinin
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance
Saunders, D.G.O. ; Breen, S. ; Win, J. ; Schornack, S. ; Hein, I. ; Bozkurt, T.O. ; Champouret, N. ; Vleeshouwers, V.G.A.A. ; Birch, P.R.J. ; Gilroy, E.M. ; Kamoun, S. - \ 2012
The Plant Cell 24 (2012)8. - ISSN 1040-4651 - p. 3420 - 3434.
nicotiana-benthamiana - plant transformation - avirulence genes - cell-death - arabidopsis - potato - activation - expression - virulence - target
Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.
A novel expression cassette for the efficient visual selection of transformed tissues in florists' chrysanthemum (Chrysanthemum morifolium Ramat.).
Mao, J. ; Stoopen, G.M. ; Jongsma, M.A. ; Wang, C.Y. - \ 2011
African journal of biotechnology 10 (2011)51. - ISSN 1684-5315 - p. 10537 - 10542.
agrobacterium-mediated transformation - green fluorescent protein - plant transformation - transgenic plants - gene-expression - stem segments - gfp - reporter - marker - leaf
Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein (GFP) reporter gene to initiate very high expression of GFP in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) was described. Based on this expression cassette, a new regeneration protocol using leaf discs as explants was developed for the Agrobacterium-mediated transformation of Chrysanthemum genotype ‘1581’, and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines. Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter. Under normal light, plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that the plastid targeted construct with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassette may allow direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant species
Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis
Li, S. ; Os, G.M.A. van; Ren, S. ; Yu, D. ; Ketelaar, T. ; Emons, A.M.C. ; Liu, C. - \ 2010
Plant Physiology 154 (2010). - ISSN 0032-0889 - p. 1819 - 1830.
lateral root emergence - exocyst complex - plasma-membrane - saccharomyces-cerevisiae - plant transformation - epithelial-cells - 19.5s particle - tip growth - pollen - thaliana
During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. In contrast to a single gene in yeast and most animals, plants have greatly increased number of EXO70 genes in their genomes, with functions very much unknown. Reverse transcription-polymerase chain reactions were performed on all 23 EXO70 genes in Arabidopsis (Arabidopsis thaliana) to examine their expression at the organ level. Cell-level expression analyses were performed using transgenic plants carrying ß-glucuronidase reporter constructs, showing that EXO70 genes are primarily expressed in potential exocytosis-active cells such as tip-growing and elongating cells, developing xylem elements, and guard cells, whereas no expression was observed in cells of mature organs such as well-developed leaves, stems, sepals, and petals. Six EXO70 genes are expressed in distinct but partially overlapping stages during microspore development and pollen germination. A mutation in one of these genes, EXO70C1 (At5g13150), led to retarded pollen tube growth and compromised male transmission. This study implies that multiplications of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis
A Bsister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis.
Folter, S. de; Shchennikova, A.V. ; Franken, J. ; Busscher, M. ; Baskar, R. ; Grossniklaus, U. ; Angenent, G.C. ; Immink, G.H. - \ 2006
The Plant Journal 47 (2006)6. - ISSN 0960-7412 - p. 934 - 946.
protein-protein interactions - floral organ identity - transcription factor family - flower development - wild-type - plant transformation - coat development - homeotic genes - thaliana - encodes
MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the 'ABC model' of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as Bsister (Bs) genes. Here, we report the isolation and characterization of a MADS-box Bs member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis Bs mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between Bs¿C¿E and Bs¿D¿E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development
Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis
Wroblewski, T. ; Finkers-Tomczak, A.M. ; Michelmore, R. - \ 2005
Plant Biotechnology Journal 3 (2005)2. - ISSN 1467-7644 - p. 259 - 273.
tumefaciens plasmid ptic58 - green fluorescent protein - cotyledonary-node cells - plant transformation - host-range - crown gall - resistance determinant - nucleotide-sequence - transgenic plants - tobacco-leaves
Agrobacterium-mediated transient assays for gene function are increasingly being used as alternatives to genetic complementation and stable transformation. However, such assays are variable and not equally successful in different plant species. We analysed a range of genetic and physiological factors affecting transient expression following agroinfiltration, and developed a protocol for efficient and routine transient assays in several plant species. Lettuce exhibited high levels of transient expression and was at least as easy to work with as Nicotiana benthamiana. Transient expression occurred in the majority of cells within the infiltrated tissue and approached 100% in some regions. High levels of transient expression were obtained in some ecotypes of Arabidopsis; however, Arabidopsis remains recalcitrant to routine, genotype-independent transient assays. Transient expression levels often exceeded those observed in stably transformed plants. The laboratory Agrobacterium tumefaciens strain C58C1 was the best strain for use in plant species that did not elicit a necrotic response to A. tumefaciens. A wild A. tumefaciens strain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species.
A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco
Mlynarova, L. ; Nap, J.P.H. - \ 2003
Transgenic Research 12 (2003)1. - ISSN 0962-8819 - p. 45 - 57.
mediated cassette exchange - matrix attachment regions - agrobacterium t-dna - transient expression - plant transformation - arabidopsis-thaliana - spacer region - gene - system - genome
To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, creINT, was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising creINT, sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with creINT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The creINT system has therefore advantages over systems with a continuously present Cre. The creINT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants