Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands.

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.

Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Terpene down-regulation in orange reveals the role of fruit aromas in mediating interactions with insect herbivores and pathogens.

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.

The monoterpene limonene in orange peels attracts pests and microorganisms.

Plant volatiles include terpenoids, which are generally involved in plant defense, repelling pests and pathogens and attracting insects for herbivore control, pollination and seed dispersal. Orange fruits accumulate the monoterpene limonene at high levels in the oil glands of their fruit peels. When limonene production was downregulated in orange fruits by the transgenic expression of a limonene synthase (CitMTSE1) in the antisense configuration, these fruits were resistant to the fungus Penicillium digitatum (Pers.) Sacc. and the bacterium Xanthomonas citri subsp. citri and were less attractive to the medfly pest Ceratitis capitata. These responses were reversed when the antisense transgenic orange fruits were treated with limonene. To gain more insight into the role of the limonene concentration in fruit responses to pests and pathogens, we attempted to overexpress CitMTSE1 in the sense configuration in transgenic orange fruits. Only slight increases in the amount of limonene were found in sense transgenic fruits, maybe due to the detrimental effect that excessive limonene accumulation would have on plant development. Collectively, these results suggest that when limonene reaches peak levels as the fruit develops, it becomes a signal for pest and pathogen attraction, which facilitate access to the fruit for pulp consumers and seed dispersers.

Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime.

Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs.

Gene stacking in 1-year-cycling APETALA1 citrus plants for a rapid evaluation of transgenic traits in reproductive tissues.

Rapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this evaluation. Through genetic transformation, juvenile periods can be significantly shortened by overexpression of flower meristem-identity genes. Transgenic APETALA1 (AP1) citrus plants behave as rapid-cycling trees, since 1-year-old seedlings promptly show precocious flowering and fruiting. By transgene stacking into these short-generation AP1 and nptII/GUS-positive plants, expression of novel transgenes could theoretically be examined as quickly as 1 year after retransformation. Establishment of the selection and regeneration conditions for the production of retransformed individuals with marker genes is detailed in this communication. Hpt and bar genes were used as the second selectable marker genes. PCR and Southern blot analyses confirmed the recovery of retransformed shoots. AP1 transcript accumulation and GUS and GFP expression were assessed in leaves, and flowers and fruit organs of rapid-cycling retransformed lines, respectively, as early as 1 year after plant generation and during three consecutive years, demonstrating that the principle of stable transgene stacking on early-fruiting transgenic trees is feasible.

Evaluation of selection strategies alternative to nptII in genetic transformation of citrus.

The neomycin phosphotransferase (nptII) selection system has proved successful in citrus transformation; however, it may be recommendable to replace it given the pressure exerted against antibiotic-resistance selectable marker genes in transgenic plants. The present work investigates three different selection alternatives, comparing them to nptII selection in two citrus genotypes, Carrizo citrange and Pineapple sweet orange. The first method used the beta-glucuronidase (uidA) reporter marker gene for selection; the second attempted to generate marker-free plants by transforming explants with a multi-auto-transformation (MAT) vector, combining an inducible R/RS-specific recombination system with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes; while the third exploited the phosphomannose isomerase (PMI)/mannose conditional positive selection system. Firstly, GUS screening of all regenerated shoots in kanamycin-free medium gave 4.3% transformation efficiency for both genotypes. Secondly, workable transformation efficiencies were also achieved with the MAT system, 7.2% for citrange and 6.7% for sweet orange. This system affords an additional advantage as it enables selectable marker genes to be used during the in vitro culture phase and later removed from the transgenic plants by inducible recombination and site-specific excision. Thirdly, the highest transformation rates were obtained with the PMI/mannose system, 30% for citrange and 13% for sweet orange, which indicates that this marker is also an excellent candidate for citrus transformation.

Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration.

Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tumefaciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows manipulation of the vegetative phase to increase the potential for transformation and regeneration without loss of competence for flowering and fruiting. To produce transgenic plants from clementine mandarin (Citrus clementina hort. ex Tanaka), we optimized the conditions of the source material both ex vitro and in vitro. Grafting of mature buds on juvenile rootstocks in the spring and preventing multiple bud sprouting by removing all but one bud permitted selection of vigorous first flushes for in vitro culture. Use of additional virulence genes from A. tumefaciens to increase transformation frequency and optimization of culture media and conditions to enhance explant cell competence for T-DNA integration and organogenesis resulted in efficient and reliable transgenic plant production. Transformed regenerants from explants, cultured in media without antibiotics, were identified by a screenable marker (either beta-glucuronidase or green fluorescent protein (GFP)), creating the possibility of generating transgenic clementine plants without antibiotic resistance marker genes. Stable integration of foreign genes was demonstrated by Southern blot analysis, and expression of these foreign genes was confirmed by detection of GFP fluorescence in leaves, floral organs and fruits of the transgenic plants.

Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus.

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination.

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. x Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.

Genetic transformation of mature citrus plants.

Most woody fruit species have long juvenile periods that drastically prolong the time required to analyze mature traits. Evaluation of characteristics related to fruits is a requisite to release any new variety into the market. Because of a decline in regenerative and transformation potential, genetic transformation procedures usually employ juvenile material as the source of plant tissue, therefore resulting in the production of juvenile plants. Direct transformation of mature material could ensure the production of adult transgenic plants, bypassing in this way the juvenile phase. Invigoration of the source adult material, establishment of adequate transformation and regeneration conditions, and acceleration of plant development through grafting allowed us to produce transgenic mature sweet orange trees flowering and bearing fruits in a short time period.

Histochemical and fluorometric assays for uidA (GUS) gene detection.

Transgenic plant production has been intimately connected to the beta-glucuronidase (uidA or GUS) gene used as a reporter marker gene. The enzyme stability and the high sensitivity and amenability of the GUS assay to qualitative (histochemical assay) and to quantitative (fluorometric or spectrophotometric assay) detection are some of the reasons that explain the extensive use of uidA gene in plant genetic transformation. Methods for uidA gene detection have been thoroughly described in the literature. The aim of this chapter is to describe the basic protocols needed for GUS detection in a plant genetic transformation laboratory.

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.