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.
Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana
Du, J. ; Rietman, H. ; Vleeshouwers, V.G.A.A. - \ 2014
Journal of Visualized Experiments (2014)83. - ISSN 1940-087X - 7 p.
late blight resistance - mediated plant transformation - phytophthora-infestans - pathogen phytophthora - disease resistance - effector proteins - gene-expression - binary vector - agrobacterium - recognition
Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.
Regeneration and transformation of Crambe abyssinica
Qi, W. ; Tinnenbroek-Capel, I.E.M. ; Schaart, J. ; Huang Bangquan, ; Cheng, J. ; Visser, R.G.F. ; Loo, E.N. van; Krens, F.A. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229 - 12 p.
gene - agrobacterium - tissue - plants
Background: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. Results: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L-1 were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L-1 kanamycin, 25 mg · L-1, then back to 10 mg · L-1) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L-1) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. Conclusions: Present research revealed the potential of using crambe meristematic tissue for genetic transformation andin vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.
Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus
Ronde, D. de; Butterbach, P.B.E. ; Lohuis, H. ; Hedil, M. ; Lent, J.W.M. van; Kormelink, R.J.M. - \ 2013
Molecular Plant Pathology 14 (2013)4. - ISSN 1464-6722 - p. 405 - 415.
mediated plant transformation - capsicum-chinense - cell-death - disease-resistance - lycopersicon-esculentum - viral suppressors - sw-5 gene - potato - tospovirus - agrobacterium
As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSsRI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the NRI/RB protein, or when NSsRB was expressed. Although NSsRI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSsRB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene
Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)
Koehorst-van Putten, H.J.J. ; Wolters, A.M.A. ; Pereira-Bertram, I.J. ; Berg, H. ; Krol, A.R. van der; Visser, R.G.F. - \ 2012
Planta 236 (2012)6. - ISSN 0032-0935 - p. 1955 - 1965.
bound-starch-synthase - storage roots - transformation - expression - gene - agrobacterium - elements - sequences - database - program
In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.
Isolation and characterization of strong gene regulatory sequences from apple, Malus x domestica
Schaart, J.G. ; Tinnenbroek, I.E.M. ; Krens, F.A. - \ 2011
Tree Genetics and Genomes 7 (2011)1. - ISSN 1614-2942 - p. 135 - 142.
rbcs-3a gene - expression - plants - elements - agrobacterium - arabidopsis - cisgenesis - resistance - promoters - tissues
For the strong expression of genes in plant tissue, the availability of specific gene regulatory sequences is desired. We cloned promoter and terminator sequences of an apple (Malus x domestica) ribulose biphosphate carboxylase small subunit gene (MdRbcS), which is known for its high expression and used gus reporter gene expression to test the regulatory activity of the isolated promoter and terminator sequences in transgenic tobacco. The MdRbcS promoter itself seemed to be less strong than the CaMV35S promoter when both used in combination with the nos terminator. However, the combination of the promoter and terminator of MdRbcS was able to drive gus to similar expression levels as the reference construct with CaMV35S promoter and nos terminator. This observation indicates the importance of the terminator sequence for gene expression. It is concluded that the combination of the MdRbcS promoter and terminator is a suitable regulatory sequence set for the expression of transgenes to a high level in plants and for intragenesis in apple specifically
Functional analysis and expression of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples
Joshi, S.G. ; Schaart, J. ; Groenwold, R. ; Jacobsen, E. ; Schouten, H.J. ; Krens, F.A. - \ 2011
Plant Molecular Biology 75 (2011)6. - ISSN 0167-4412 - p. 579 - 591.
receptor-like genes - real-time pcr - venturia-inaequalis - vf gene - plants - sequences - agrobacterium - promoters - linkage - cluster
Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRTPCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.
Anthocyanin production as a potential visual selection marker during plant transformation
Kortstee, A.J. ; Khan, S.A. ; Helderman, C.M. ; Trindade, L.M. ; Wu, Y. ; Visser, R.G.F. ; Brendolise, C. ; Allan, A.C. ; Schouten, H.J. ; Jacobsen, E. - \ 2011
Transgenic Research 20 (2011)6. - ISSN 0962-8819 - p. 1253 - 1264.
transcription factor - dna transformation - transgenic plants - expression - genes - cultures - agrobacterium - biosynthesis - regeneration - efficiency
A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance
Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines
Krens, F.A. ; Schaart, J.G. ; Groenwold, R. ; Walraven, A.E.J. ; Hesselink, T. ; Thissen, J.T.N.M. - \ 2011
Transgenic Research 20 (2011)5. - ISSN 0962-8819 - p. 1113 - 1123.
receptor-like genes - scab resistance - venturia-inaequalis - expression - thionins - vf - transformation - agrobacterium - plants - wheat
Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable
Delayed senescence in cauliflower transformed with an autoregulated isopentenyl transferase gene
Nguyen, K.H. ; Jordi, W.J.R.M. ; Dun, K. ; Schepers, F. ; Davelaar, E. ; Stoopen, G.M. ; Dix, P.J. ; Kane, E.J. - \ 2008
International Journal of Plant Sciences 169 (2008)3. - ISSN 1058-5893 - p. 339 - 347.
leaf senescence - tobacco plants - cytokinin - agrobacterium - expression - cultures
Cauliflower (Brassica oleracea var. botrytis) was transformed, using Agrobacterium tumefaciens, with an autoregulated isopentenyl transferase (ipt) gene under the control of a senescence-associated gene promoter, pSAG12, isolated from Arabidopsis thaliana. The effect of introducing this chimeric construct on cytokinin (CK) content, chlorophyll retention, and plant morphology and development were investigated. A range of CK and chlorophyll contents was found among the individual primary transformants. Progeny were studied from one of the primary transformed lines that did not have elevated cytokinin content and was phenotypically similar to the parent line but displayed delayed leaf senescence. The pSAG12:ipt gene was inherited in a Mendelian manner, and the effect of this gene on senescence-related parameters was observed in a number of the progeny. While the pSAG12:ipt progeny did exhibit delayed leaf senescence, it was accompanied by undesirable agronomic traits, including less synchronous curd initiation, smaller curd size, and greater susceptibility to fungal infection.
Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis
Kudla, U. ; Milac, A. ; Qin Ling, ; Overmars, H.A. ; Roze, E.H.A. ; Holterman, M.H.M. ; Petrescu, A.J. ; Goverse, A. ; Bakker, J. ; Helder, J. ; Smant, G. - \ 2007
Molecular Plant Pathology 8 (2007)3. - ISSN 1464-6722 - p. 293 - 305.
subventral esophageal glands - protein secondary structure - heterodera-glycines - developmental expression - erwinia-chrysanthemi - structure prediction - new-generation - agrobacterium - identification - transformation
The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca2+ is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes
Agroinjection of Tomato Fruits : a Tool for Rapid Functional Analysis of Transgenes Directly in Fruit
Orzaéz Calatayud, D.V. ; Mirabel, S. ; Wieland, W.H. ; Granell, A. - \ 2006
Plant Physiology 140 (2006)1. - ISSN 0032-0889 - p. 3 - 11.
transient gene-expression - b-subunit - virus - agrobacterium - protein - leaves - arabidopsis - synthase - mutant - plants
Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven -glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.
Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)
Zheng Sijun, S.J. ; Henken, B. ; Maagd, R.A. de; Purwito, A. ; Krens, F.A. ; Kik, C. - \ 2005
Transgenic Research 14 (2005)3. - ISSN 0962-8819 - p. 261 - 272.
tumefaciens-mediated transformation - synthetic sex-pheromone - delta-endotoxin - communication disruption - plant-regeneration - domain iii - agrobacterium - lepidoptera - noctuidae - dna
Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot
Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene
Schaart, J.G. ; Krens, F.A. ; Pelgrom, K.T.B. ; Mendes, O. ; Rouwendal, G.J.A. - \ 2004
Plant Biotechnology Journal 2 (2004)3. - ISSN 1467-7644 - p. 233 - 240.
cytosine deaminase - glucocorticoid receptor - transformation vector - negative selection - bacterial gene - expression - agrobacterium - regeneration - system - frequency
Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.
An ancient R gene from Solanum bulbocastanum confers broad-spectrum resistance to late Phytophthora infestans in cultivated potato and tomato
Vossen, E.A.G. van der; Sikkema, A. ; Lintel Hekkert, B. te; Gross, J. ; Stevens, P. ; Muskens, M. ; Wouters, T.C.A.E. ; Pereira, A.B. ; Stiekema, W.J. ; Allefs, S. - \ 2003
The Plant Journal 36 (2003)6. - ISSN 0960-7412 - p. 867 - 882.
nucleotide-binding site - disease resistance - late blight - avirulence gene - cell-death - virus-resistance - s-bulbocastanum - arabidopsis - locus - agrobacterium
Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection
Tissue-specific expression of the beta-glucuronidase reporter gene in transgenic strawberry (Fragaria Xananassa) plants
Schaart, J.G. ; Salentijn, E.M.J. ; Krens, F.A. - \ 2002
Plant Cell Reports 21 (2002)4. - ISSN 0721-7714 - p. 313 - 319.
mediated transformation - agrobacterium - ethylene - fruit
The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter