Nova Biotechnol Chim (2017) 16(1): 20-25 DOI: 10.1515/nbec-2017-0003  Corresponding author: martincova21@uniba.sk Nova Biotechnologica et Chimica Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton Peter Kaštiera, Michaela Martinčováa,, Roderik Fialab and Alžbeta Blehováa a Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 3278/6, Bratislava, SK-841 04, Slovak Republic b Institute of Botany, Plant Science and Biodiversity Centre SAS, Dúbravská cesta 9, Bratislava, SK-845 23, Slovak Republic Article info Article history: Received: 22nd March 2017 Accepted: 16th May 2017 Keywords: Actin Agrobacterium tumefaciens Cuscuta europaea gfp Parasitic plants Transient transformation Abstract Dodder (Cuscuta) species cause severe agricultural damage in many countries throughout the world. To establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. For these efforts genetic modification would represent a powerful tool. Here we report on Agrobacterium- mediated transformation of dodder using green fluorescent protein (GFP) fused to actin-binding protein as a vital marker. Since the shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. The transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. Transient expression of GFP appears to be a suitable method for studying Cuscuta development through cytoskeleton organisation that is presently largely unexplored.  University of SS. Cyril and Methodius in Trnava Introduction Parasitic Cuscuta (dodder) genus (Convolvulaceae) contains 194 species divided into four subgenera, namely Monogynella, Cuscuta, Grammica and Pachystigma (Costea et al. 2015). They are spread nearly worldwide (García et al. 2014). Dodders are twining herbs with reduced vegetative organs (leaves are minute scales). Dodder species cause severe agricultural damage in many countries throughout the world resulting in yield decrease of important crops such as tomatoes and potatoes (Heide-Jørgensen 2011). In spite of their agricultural importance, many questions remain unanswered and an efficient transformation system would greatly accelerate the progress in understanding host-parasite relationships. Research on mechanisms of haustorium formation as of key organ necessary for dodder survival is a prerequisite for developing efficient strategies to control its development and spreading. Understanding the interaction between host plants and parasitic dodders at the ultrastructural and molecular levels may generate knowledge applicable to control plant-parasitic interactions and/or engineer dodder-resistant plants. Though efficient transformation protocols have been described for dodder calli (Borsics et al. 2002; Švubová and Blehová 2013), these have never resulted in regeneration of whole plants (Ichihashi et al. 2015). Here we report on transformation of excised dodder shoot explants via Agrobacterium that resulted in transient expression of the vital marker gfp gene. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:36 UTC mailto:martincova21@uniba.sk Nova Biotechnol Chim (2017) 16(1): 20-25 21 To study the transgenic nature and development of transformed tissue we used a GFP protein fused in frame to a protein that binds to actin. Actin as a component of the cytoskeleton coordinates numerous cellular processes required for plant development, including cell division and cell expansion (Wasteneys and Ambrose 2009), targeted-vesicle transport to establish hormone gradients (Muday 2000), polarity determination to direct localized wall biosynthesis (Baluška et al. 2003), and organelle positioning in response to environmental cues (Šamaj et al. 2006). In Cuscuta species, the presence of α-tubulin, though in quantities lower than in roots, has been associated with the degradation processes of the root-like structures (Sherman et al. 2008). Further, indirect immunolocalization of actin as well as tubulin has indicated important role in prehaustorial cells in C. europaea (Kaštier et al. 2015). The techniques used in those studies, however, do not allow to study the dynamic changes in cytoskeleton organization in real-time in living cells, therefore would largely benefit from a use of a vital marker like GFP (Švubová and Blehová 2013; Blehová et al. 2015). Experimental Plant material Parasitic dodder seeds (C. europaea) were collected in Ivanka pri Dunaji (2014, Slovakia, latitude 48°19´, longitude 17°22´) and their host plant was Urtica dioica. The seeds were scarified at 4 °C and then treated with concentrated H2SO4 for 60 min at room temperature in order to disrupt the seed coat integration. Acid was washed-off by sterile distilled water. The seeds were then sterilized with 5 % Savo (0.24 % NaClO) solution for 15 min and thoroughly washed with sterile distilled water. Dodders were cultivated in vitro on MS medium (Murashige and Skoog 1962) containing agar (7 g/L), pH 5.8, in the growth chamber (16/8 h long-day photoperiod) at 23±2 oC, 100 μmol/m2s PAR. Bacterial strains and cultivation conditions Agrobacterium tumefaciens LBA4404 carrying the plasmid pCB302 (Voigt et al. 2005) was used for dodder transformation. The T-DNA of the pCB302 contained a single GFP fused with N-terminus of Arabidopsis thaliana fimbrin actin-binding domain that is driven by constitutive CaMV 35S promoter. As a plant selection marker, the neomycin phosphotransferase II gene (nptII) was used under control of the nos promoter. A. tumefaciens/pCB302 was cultivated on LB (Luria-Bertani) medium, pH 6.9. Bacterial suspension (300 µL) and 100 mg/L of kanamycin were added into a liquid LB medium and cultivated for 12 hours on a shaker (SHAKER DOS-20L; 120 rpm) in the dark at 23 °C. Bacterial transformation Segments of C. europaea stems (7–8 mm shoot apices) were infected by A. tumefaciens/pCB302 in the liquid LB medium for 10 min and subsequently co-cultivated on MS medium without antibiotics overnight in the dark at 23±2 °C. Infected segments were washed by a liquid MS medium containing cefotaxime (200 mg/L). After drying on a sterile paper, explants were grown on a selective MS medium containing agar and antibiotics – kanamycin (100 mg/L) and cefotaxime (200 mg/L). They were inoculated on fresh selective media in one week sub-cultivation intervals. To monitor plant growth, shoot lengths were measured every week for six weeks of in vitro cultivation. Microscopy of dodders Light microscopy of C. europaea seedlings was carried out using a stereomicroscope (Leica M165 FC). GFP positive dodder stem segments were examined by a confocal microscope(Olympus FV1000) at an excitation wavelength 488 nm. GFP fluorescence and plastid autofluorescence were detected using 505 – 550 nm and 655 – 755 nm barrier emission filters, respectively. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:36 UTC Nova Biotechnol Chim (2017) 16(1): 20-25 22 Results and Discussion Technical advances in transformation techniques have facilitated research in parasitic plants, too. Transgenic dodder calli or roots derived from A. rhizogenes-mediated transformations still were able to form lateral haustoria that penetrated the host (Fernández-Aparicio et al. 2011; Ishida et al. 2011). However, stable and heritable transformation of Cuscuta plants, enabling molecular genetics studies of these species, have not been established yet, in part due to challenges in developing transformation protocols, but also because regeneration of a parasitic organism in vitro is difficult (Borsics et al. 2002; Švubová and Blehová 2013). Since the C. europaea seedlings germinate as cotyledonless and leafless shoots with ephemeral root-like structure, transformation of shoot apex can be used as an alternative for Agrobacterium-mediated transformation of monocotyledonous crop species (Gould et al. 1991; this study). Fig. 1. Growth and development of C. europaea seedlings in vitro. A. Germinating dodder seeds on MS medium on 1st day after germination. B. Germinating seed with tuberous radicle and root tip (circle). C. Detail of the root tip (circle) with hairs. D. Dodder leafless stem with shoot apex (rectangle) on 7th day after germination. E. Early degradation (7th day after germination; asterisks) of root-like structure with visible xylem (arrow). F. Detail of the degrading root- like structure (asterisks) on 7th day after germination. G. Progressing degradation (asterisks) of the root-like structure on 14th day after germination, xylem (arrow). H. Detailed view on root-like structure in the late stage of degradation (asterisks) on 14th day after germination. Scale bars: A = 2 cm; B, D, E, G = 2 mm; C, F, H = 0.5 mm. Dodder seeds were transferred to in vitro conditions. According to some authors, stratification by cold and scarification are essential for their successful germination (Švubová and Blehová 2013; Furuhashi et al. 2016). Variability of seed coat structure among different dodder species determines their germination characteristics impacting adaptation to natural environment (Behdarvandi et al. 2015). The seeds started to germinate 2–3 days after sowing (Fig. 1A). A visibly swollen radicle of C. europaea emerged from the seeds as the first structure (Fig. 1A and B) and this was followed by the emergence of a stem (Fig. 1D). Since the first day after germination, differentiation of root hairs at the radicle tip was observed (Fig. 1C). In contrast, in the case of C. campestris, the bulges resembling root hairs are absent and the surface of the radicle-like structure is smooth (Lyshede 1985). According to Sherman et al. (2008), these root hairs do not elongate and have a different ultrastructure compared to the root hairs of typical dicots. In addition, the root-like structure of dodder is considered to be a remnant of the true shoot that was significantly modified during evolution (Sherman et al. 2008). For example, in C. europaea it lacks both root cap and root apical meristem (Fig. 1B and C) as observed by other authors (Lyshede 1986; Lee et al. 2000; Behdarvandi et al. 2015). Several days after germination this root-like organ degrades through a senescence-like process from apex towards the stem base (Fig. 1E – H), possibly due to low abundance of microtubules in its cells and lack of mitotic division (Sherman et al. 2008). Nevertheless, this structure is still capable of interacting with microorganisms, therefore Behdarvandi et al. (2015) consider this organ to be a modification of true root. Moreover, xylem strands with non-lignified cell walls were clearly visible even under a light microscope (Fig. 1E and G) indicating to functional translocation of some nutrients into the rapidly growing shoot (Sherman et al. 2008). The development of C. europaea plants shows a remarkable degree of plasticity to a large extent attributed to shoot apical meristem. In contrast to the pale root-like structure C. europaea stem tips are green (Fig. 1B and D). Several photosynthesis- related proteins have been described in dodder stems (e.g. RbcL, THF1, D1) indicating that Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:36 UTC Nova Biotechnol Chim (2017) 16(1): 20-25 23 Fig.2. Lengths of dodder shoot explants during 35 days of in vitro cultivation. GFP positive (full line) shoot explants transformed by A. tumefaciens/pCB302. Dot dotted line represents the length of control shoot explants. dodders in this non-parasitic developmental stage are photosynthetically active (Švubová et al. 2013). In addition, they contain much more actin and microtubule elements compared to roots (Sherman et al. 2008) enabling rapid growth of dodder shoot explants on hormone free MS media (Fig. 2). Dynamic reorganization of the cytoskeleton in combination with photosynthetic activity of young dodder seedlings is likely responsible for ability to survive without host plants for up to 3 weeks (Heide-Jørgensen 2008). Transformation of excised shoot explants was performed using the construct pCB302 which has successfully been used for actin visualization in transgenic Arabidopsis plants (Voigt et al. 2005; Wang et al. 2007). After transformation, dodder tissue was cultivated and its growth was monitored. We observed strong GFP fluorescence, especially in the shoot tips (Fig. 3A). Since the marker protein (GFP) was fused to actin-binding protein, higher magnification images of cells in the transformed shoot’s apical zone, revealed a network of fine F-actin surrounding the nuclei (Fig. 3B) as described previously for other plant species (Chiu et al. 1996; Wang et al. 2007). However, Fig. 3. GFP fluorescence of C. europaea shoot explants analysed by confocal microscopy. A) GFP positive dodder shoots on 7th day after transformation, arrow indicates stronger GFP positive signal in the meristematic region of the shoot apex. B) GFP fluorescence of the stem tip epidermal cells. C) The positive signal in the cells of elongation zone on 7th day after transformation. D) The elongating cells with lowered GFP fluorescence on 14th day after transformation. E) Autofluorescence of GFP negative dodder stem on 35th day after transformation. F) Autofluorescence of control stem of non-transformed C. europaea. Scale bars: A, F = 200 µm; B, C, D = 50 µm; E = 25 µm. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:36 UTC Nova Biotechnol Chim (2017) 16(1): 20-25 24 in the distal elongation zone the GFP fluorescence turned considerably weaker (Fig. 3C–E). This observation is consistent with significantly reduced growth of the transformed dodder stems in comparison with the control explants (Fig. 2). Nevertheless, the exact reason for this phenomenon is unclear and might also include problems with the transformation procedure (e.g. gradual degradation of un-incorporated T-DNA, gradual silencing of the transgene expression, recalcitrant dodder genotype etc.) or failing of the binding of the fusion protein in the tissue, or both. Nevertheless, fluorescence of the transgene product in dodder tissue (cultivated in presence of selection agent for 7–14 days), reflects to transient transgene expression that allows for non-invasive observation of cytoskeleton organisation in developing shoot tissue of the parasite. The presence and activity of the transgene in transformed dodder tissue needs to be verified in more detail at molecular level. Further, since dodder shoots appeared as GFP positive only for a relatively short period of time, further optimization of the transformation procedure using e.g. sonication followed by vacuum treatment (Fernández-Aparicio et al. 2011). Enhancement of assays for transient gene expression will probably involve optimization of factors promoting T-DNA transfer e.g. over-expression of bacterial virulent virE and virG genes can enhance transient expression (Wroblewski et al. 2005). Also virD that probably plays a major role in targeting T- strands to the nucleus through α importins localized in nuclear envelope (Gelvin 2012). In addition, it is also beneficial to apply Agrobacterium strains that carry specific P19 helper plasmids as silencing suppressors (Voinnet et al. 2003; Canto et al. 2006), what will be a challenge for future. Conclusions The shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, therefore shoot apex was used here for genetic transformation via A. tumefaciens. The fluorescence pattern of the marker gene product suggests only a transient transformation. Fusion of GFP with actin-binding element appeared as feasible for not only confirming the presence of transgene product in host tissue but also for studying the organization and of cytoskeleton in the vital dodder tissue. 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