Henryk Flachowsky

The development of a cisgenic apple plant

Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g. antibiotic or herbicide resistance) in the final product and the fact that the inserted gene(s) derive from organisms sexually compatible with the target crop should rise less environmental concerns and increase consumers acceptance. Here we report the generation of a cisgenic apple plant by inserting the endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated transformation combined with a positive and negative selection system and a chemically inducible recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for detailed investigation and were shown to carry a single T-DNA insertion and express the target gene HcrVf2. This is the first report of the generation of a true cisgenic plant.

Maize Lc transcription factor enhances biosynthesis of anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple (Malus domestica Borkh.)

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple (Malus domestica Borkh.) by overexpression of the maize (Zea mays L.) leaf colour (Lc) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens-mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc-transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc-transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.

Shift in polyphenol profile and sublethal phenotype caused by silencing of anthocyanidin synthase in apple (Malus sp.).

We have investigated the consequences of blocking anthocyanin biosynthesis by silencing a key enzyme, anthocyanidin synthase, in transgenic plants of a red-leaved apple cultivar. This is complementary to a previous study of induction of anthocyanin biosynthesis by overexpressing a heterologous transcription factor. Analysis of these opposite phenotypes allows one to study anthocyanin functions in apple and to test the influence of the genetic manipulation on other, related metabolites. As expected, anthocyanin biosynthesis was almost completely blocked and this was accompanied by a shift in the profile of flavonoids and related polyphenols. Most interestingly, a rise in epicatechin was found. A severe reduction of viability by necrotic leaf lesions was also observed, suggesting an essential function of anthocyanins in apple.

The MdTFL1 gene of apple (Malus × domestica Borkh.) reduces vegetative growth and generation time

TFL1 is known as a floral repressor in Arabidopsis thaliana (L.) Heynh. In apple there are two TFL1 homologs, MdTFL1-1 and MdTFL1-2. The MdTFL1-1 gene was silenced in transgenic clones expressing a hairpin gene construct of a 323 bp fragment of MdTFL1-1. The hairpin gene construct was transferred to three different apple genotypes. Of 22 transgenic clones, 21 showed a significant reduction in MdTFL1-1 mRNA expression. Precocious flowering was obtained for 20 clones, which flowered already during in vitro cultivation. Nineteen clones could successfully be transferred to the greenhouse where 18 of them flowered within a few weeks followed by the death or at least a strongly inhibited vegetative growth of the plant. Most of the transgenic flowers developed abnormally. Results obtained on greenhouse-grown plants of the transgenic clones and transgenic seedlings clearly demonstrated the major role of MdTFL1 genes in maintaining the vegetative growth as prerequisite for a perennial lifecycle. It was shown that MdTFL1 dsRNAi promotes a life history similar to annual plants. Preliminary results obtained from grafting experiments with non-transgenic scions grafted onto MdTFL1 dsRNAi transgenic rootstocks indicated that the flower-inducing signal obtained after silencing of MdTFL1 genes seems not to be graft-transmissible.

Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants

The generation of transgenic apple plants relies on the molecular analysis of transgene integration and expression based on polymerase chain reaction (PCR) analysis, blotting techniques and enzymatic assays on vitro leaves of putative transgenic regenerates. In order to assess the uniformity and the stability of transfer DNA (T-DNA) integration and gene expression, we studied 26 transgenic apple lines carrying the attacin E gene from Hyalophora cecropia , the β-glucuronidase gene, and the nptII gene. Plants were evaluated using standard molecular techniques, such as PCR, Southern blot, reverse transcription PCR (RT-PCR) and Enzyme Linked Immunosorbent Assay (ELISA), and propagated in vitro on non-selective antibiotic-free media for four years to mimic natural conditions in the field. In some T-lines transgene integration and expression did not remain stable; differences were also found between distinct plants of a single T-line. Individual plants with partially or completely silenced transgenes were identified as well as plants with non-detectable T-DNA. Several lines appeared chimeric or partially silenced. Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or cells with silenced transgenes. This should be taken into consideration, especially in the case of vegetatively propagated trees, where non-transformed or silenced plant parts could mistakenly be used as propagation material.

Gene-for-gene relationship in the host–pathogen system Malus × robusta 5–Erwinia amylovora

Fire blight is a destructive bacterial disease caused by Erwinia amylovora affecting plants in the family Rosaceae, including apple. Host resistance to fire blight is present mainly in accessions of Malus spp. and is thought to be quantitative in this pathosystem. In this study we analyzed the importance of the E. amylovora effector avrRpt2(EA) , a homolog of Pseudomonas syringae avrRpt2, for resistance of Malus × robusta 5 (Mr5). The deletion mutant E. amylovora Ea1189ΔavrRpt2(EA) was able to overcome the fire blight resistance of Mr5. One single nucleotide polymorphism (SNP), resulting in an exchange of cysteine to serine in the encoded protein, was detected in avrRpt2(EA) of several Erwinia strains differing in virulence to Mr5. E. amylovora strains encoding serine (S-allele) were able to overcome resistance of Mr5, whereas strains encoding cysteine (C-allele) were not. Allele specificity was also observed in a coexpression assay with Arabidopsis thaliana RIN4 in Nicotiana benthamiana. A homolog of RIN4 has been detected and isolated in Mr5. These results suggest a system similar to the interaction of RPS2 from A. thaliana and AvrRpt2 from P. syringae with RIN4 as guard. Our data are suggestive of a gene-for-gene relationship for the host-pathogen system Mr5 and E. amylovora.

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.

RNA-Mediated Gene Silencing Signals Are Not Graft Transmissible from the Rootstock to the Scion in Greenhouse-Grown Apple Plants Malus sp.

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.

TERMINAL FLOWER1 is a breeding target for a novel everbearing trait and tailored flowering responses in cultivated strawberry (Fragaria × ananassa Duch.)

Summary The effects of daylength and temperature on flowering of the cultivated octoploid strawberry (Fragaria × ananassa Duch.) have been studied extensively at the physiological level, but information on the molecular pathways controlling flowering in the species is scarce. The flowering pathway has been studied at the molecular level in the diploid short‐day woodland strawberry (F. vesca L.), in which the FLOWERING LOCUS T1 (FvFT1)–SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FvSOC1)–TERMINAL FLOWER1 (FvTFL1) pathway is essential for the correct timing of flowering. In this work, we show by transgenic approach that the silencing of the floral repressor FaTFL1 in the octoploid short‐day cultivar ‘Elsanta’ is sufficient to induce perpetual flowering under long days without direct changes in vegetative reproduction. We also demonstrate that although the genes FaFT1 and FaSOC1 show similar expression patterns in different cultivars, the regulation of FaTFL1 varies widely from cultivar to cultivar and is correlated with floral induction, indicating that the transcription of FaTFL1 occurs at least partially independently of the FaFT1–FaSOC1 module. Our results indicate that changing the expression patterns of FaTFL1 through biotechnological or conventional breeding approaches could result in strawberries with specific flowering and runnering characteristics including new types of everbearing cultivars.