Plant Mol Biol Rep (2010) 28:185–192 DOI 10.1007/s11105-009-0141-0
Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp. citri Suane Coutinho Cardoso & Janaynna Magalhães Barbosa-Mendes & Raquel Luciana Boscariol-Camargo & Rock Seille Carlos Christiano & Armando Bergamin Filho & Maria Lúcia Carneiro Vieira & Beatriz Madalena Januzzi Mendes & Francisco de Assis Alves Mourão Filho
Published online: 4 September 2009 # Springer-Verlag 2009
Abstract Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic S. C. Cardoso : R. S. C. Christiano : A. B. Filho : M. L. C. Vieira : F. A. A. Mourão Filho (*) Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, 13418-900 Piracicaba, SP, Brazil e-mail:
[email protected] J. M. Barbosa-Mendes : R. L. Boscariol-Camargo : B. M. J. Mendes Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, 13409-970 Piracicaba, SP, Brazil Present Address: R. L. Boscariol-Camargo Centro APTA Citros Sylvio Moreira, 13490-970 Cordeirópolis, SP, Brazil
lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and sprayinoculated with an Xcc suspension (106 cfu mL−1). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (nontransgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16–0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen. Keywords Agrobacterium tumefaciens . Antimicrobial peptide . Asian citrus canker . Disease resistance . Genetic improvement . Transformation
Introduction Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri (Xcc) (Schaad et al. 2006) is one of the most important disease problems in the main citrus-producing areas in the world. Xcc infects leaves, stems, and fruits,
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causing defoliation and premature fruit drop (Graham et al. 2004). Therefore, ACC may restrict nursery material and fruit commercialization to areas where the pathogen is not present (Gottwald et al. 2001). Moreover, disease susceptibility and symptom expression in sweet orange can vary among cultivars (Gottwald et al. 2002). Currently, there are endemic areas where ACC management relies on integrated measures, including production of disease-free nursery stock, copper sprays, reduction of pathogen spread (Graham et al. 2006; Balogh et al. 2008), and areas where the pathogen is kept under control through exclusion and eradication programs. In both cases, the replacement of susceptible citrus cultivars with fieldresistant material is one of the main points for success (Gottwald et al. 2002). Genetic engineering has been considered a powerful tool in crop protection against insect pests, weeds, and pathogens, including fungi, bacteria, and virus (Collinge et al. 2008). This technology allows the introduction of specific genes into the plant genome, maintaining the genetic background of the cultivar, which is especially desirable in perennial crops such as citrus that have limitations in conventional breeding (Omar et al. 2007). Among the strategies for the genetic engineering of plants for disease resistance, the use of antimicrobial peptides constitutively expressed in plant tissues has been suggested. Attacin, cecropin, and sarcotoxin genes, derived from different insect genomes, have been introduced in different plant species with promising results (Reynoird et al. 1999; Ko et al. 2000, 2002; Bespalhok Filho et al. 2001). Transgenic potato (Solanum tuberosum L.) expressing cecropin and tobacco (Nicotiana tabacum L.) expressing shiva-1 genes (a cecropin synthetic analog) showed an increase in resistance to Ralstonia solanacearum Smith Yabuuchi et al. (Montanelli and Nascari 1991; Jaynes et al. 1993). Attacins, in general, including A, B, C, and D basic forms and E and F acidic forms (Hultmark et al. 1983), act on the outer membrane of Gram-negative bacteria, leading to changes in its permeability (Engstrom et al. 1984; Carlsson et al. 1998). Attacin A, isolated from Trichoplusia ni (Kang et al. 1996), belongs to one of the largest classes of antimicrobial peptides, with approximately 20 kDa (Lazzaro and Clark 2001). The gene that codes for this peptide has 1,601 bp with two introns and a signal peptide (Kang et al. 1996). As it occurs in insect cells, the antimicrobial peptide is secreted to the apoplast, outside of the plant cell (Boscariol et al. 2006). Attacin genes (att) have been used in fruit genetic transformation and have led to an increase in resistance against bacterial diseases. Transgenic apple [Malus×sylvestris (L.) Mill var. domestica (Borkh.) Mansf.] expressing the basic attA form (Norelli and Aldwinckle 1993) as well
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as transgenic apple and pear (Pyrus communis L.) expressing the acidic attE form (Norelli et al. 1994; Ko et al. 2002; Reynoird et al. 1999) showed a reduced susceptibility to fire blight Erwinia amylovora (Burr) Winslow et al. Specific research carried out in citrus revealed a reduction of 55–60% in ACC disease severity in attA Hamlin sweet orange transgenic plants (Citrus sinensis L. Osbeck), a cultivar highly susceptible to Xcc (Boscariol et al. 2006). These promising results led us to hypothesize how transgenic plants of other sweet orange cultivars carrying attA gene would behave against ACC pathogen. Pera is the most important cultivar in Brazil, with more than 40% of the area cultivated with sweet oranges in that country. It is considered a mid-season, mid- to highyielding cultivar. Natal sweet orange is a late-maturing cultivar, similar to Valencia, with great economical importance in Southern Brazil. Valencia sweet orange is a high-yielding, late-maturing cultivar, planted in the main citrus-growing regions in the world. Fruits of this cultivar can be used for juice processing or commercialized in the fresh market (Saunt 1990). Therefore, the objective of this study was to genetically transform Natal, Pera, and Valencia sweet orange cultivars (C. sinensis L. Osbeck) with attA gene and evaluate their resistance to Xcc.
Materials and Methods Genetic Transformation and Molecular Characterization of the Regenerants The gene construct utilized for genetic transformation consisted of the pCattA 2300 plasmid binary vector containing the 35S-35S-attA-35ST expression cassette that codes for the attacin A antimicrobial peptide and a native signal peptide responsible for directing the insect protein to the extracellular space (Boscariol et al. 2006). This expression cassette was cloned into pCambia 2300 binary vector (CAMBIA, Canberra, Australia), which carries the nptII selection gene, and introduced into Agrobacterium tumefaciens EHA 105 by the freeze–thaw method (Holsters et al. 1978). Genetic transformation of Natal, Pera, and Valencia sweet orange cultivars (C. sinensis L. Osbeck) was carried out using epicotyl segments as explants collected from in vitro-germinated seedlings (Boscariol et al. 2003). The protocols for A. tumefaciens cultivation, explant selection and preparation, and genetic transformation have been previously described (Mendes et al. 2002; Almeida et al. 2003). Adventitious buds regenerated were analyzed by polymerase chain reaction (PCR) to identify putative transgenic plants (Boscariol et al. 2006) and in vitro grafted
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onto Carrizo citrange (C. sinensis×Poncirus trifoliata). The developed plants were acclimatized in a greenhouse (28± 2°C, 80±10% RU). Genetic transformation efficiency was expressed as the percentage of transgenic (PCR-positive) plants in relation to the total number of explants exposed to A. tumefaciens. Southern blot analysis was applied to confirm gene integration into the plant genome. Genomic DNA of acclimatized plants and of non-transgenic plants (negative control) was isolated from fully expanded leaves by the CTAB method (Doyle and Doyle 1990). DNA samples (60 µg) were digested with HindIII restriction enzyme, which cuts the T-DNA once outside the gene area, on a 1% agarose gel separated by electrophoresis (1.2 V/cm) and transferred to a nylon membrane (Hybond-N+, Amersham Biosciences, Buckinghamshire, UK). A fragment of 350 bp from the attA gene was amplified by PCR, fluoresceinlabeled (Gene Images™ Random Primer Libelling Module, Amersham Biosciences), and used as a probe. Transcription of the attA gene was verified by Northern blot analysis. Total leaf RNA from transgenic and nontransgenic (negative control) plants was extracted using the TRIzol® reagent protocol (Invitrogen Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. Electrophoresis was carried out on a 1% denaturing gel with 30 μg of RNA from each sample. The separated RNAs were transferred to a nylon membrane (Hybond-N+, Amersham Biosciences), and transgene mRNA transcripts were detected by hybridization at 60°C with the same fluorescein-labeled probe described for Southern blot analysis. Propagation of Transgenic Plants and Evaluation for Resistance to Xcc Twenty-three transgenic lines, including six of Natal, seven of Pera, and ten of Valencia sweet oranges, were selected for propagation and evaluation for resistance to Xcc. For propagation, 40 well-developed buds of each transgenic line were grafted onto Rangpur lime (Citrus limonia Osbeck) rootstock plants cultivated in small tubes (19.5× 5.0 cm) filled with pinus bark commercial potting mix (Rendmax® Eucatex®, Paulínia, Brazil). Buds of nontransgenic plants were also propagated and used as negative controls. Budded plants were cultivated in a screenhouse, and scions were grown in a single stem system for approximately 2 months. Seven to ten plants of each transgenic line and the control were selected for adequate stomatal bacteria penetration (Xcc inoculation) based on leaf phenology (Viloria et al. 2004; Boscariol et al. 2006). Xcc bacteria strain IBSBF 1421, selected for inoculation, was cultured in nutrient agar medium for 48 h at 27±1°C, suspended in sterile distilled water, and concentration-
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adjusted to 106 cfu mL−1 using a colorimeter at 480 nm. Experimental plants were individually spray-inoculated on both sides of the young leaves with a 106 cfu mL−1 Xcc suspension and covered with a clear plastic bag for 72 h in order to keep high humidity (Dalla Pria et al. 2006; Pavan et al. 2007). Plants were then transferred to a growth chamber (Conviron® model PGW 36, Winnipeg, Canada) with restricted access due to biosafety regulations, in a 12-h photoperiod, at 28°C. Disease severity was evaluated 30 days after inoculation in ten to 14 leaves of each plant. The images of two young leaves per plant (seven to ten plants of each transgenic line) were digitized, and leaf diseased area was calculated using QUANT 1.0 software (Vale et al. 2001). Selected leaves corresponded to the second leaf pair below the apical meristem, expanded by two thirds, which are considered more susceptible to Xcc infection (Gottwald and Graham 1992; Graham et al. 1992). Data collected included the percentage of leaf diseased area (LDA) and resistance to Xcc expressed as percentage of LDA related to the control (non-transgenic cultivar). The experiment followed a completely randomized design and was repeated three times. Data were submitted to analysis of variance, and means of the control for each cultivar were individually compared with means of each correspondent transgenic line by Dunnett’s test (P
