Vicente J. Febres

Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes

Abstract. Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3′ end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Transgenic resistance to Citrus tristeza virus in grapefruit.

Grapefruit (Citrus paradisi) transgenic plants transformed with a variety of constructs derived from the Citrus tristeza virus (CTV) genome were tested for their resistance to the virus. Most transgenic lines were susceptible (27 lines), a few were partially resistant (6 lines) and only one line, transformed with the 3′ end of CTV was resistant. Transgene expression levels and siRNA accumulation were determined to identify whether the resistance observed was RNA-mediated. The responses were varied. At least one resistant plant from a partially resistant line showed no steady-state transgene mRNA, siRNA accumulation and no viral RNA, implicating posttranscriptional gene silencing (PTGS) as the mechanism of resistance. The most resistant line showed no transgene mRNA accumulation and promoter methylation of cytosines in all contexts, the hallmark of RNA-directed DNA methylation and transcriptional gene silencing (TGS). The variety of responses, even among clonally propagated plants, is unexplained but is not unique to citrus. The genetics of CTV, host response or other factors may be responsible for this variability.

Decreasing unpalatable flavonoid components in Citrus: the effect of transformation construct

Citrus species accumulate large quantities of flavanone glycosides in their leaves and fruit. The physiological role(s) of these compounds in citrus plants are unknown, but they have been documented to benefit human health upon consumption. Flavanone rutinosides are tasteless, whereas flavanone neohesperidosides, such as naringin, give a bitter taste to fruit and fruit juice products, reducing their palatability. In an effort to alter the types and levels of flavanone neohesperidosides in citrus, an Agrobacterium-mediated genetic transformation approach was employed. Citrus paradisi Macf. (grapefruit) epicotyl stem segments were transformed with sense (S) and antisense (AS) constructs of the target genes chalcone synthase (CHS) and chalcone isomerase (CHI), whose products catalyze the first two steps in the flavonoid biosynthetic pathway. Transformation with each of the individual constructs led to a different and unpredictable combination of viability, phenotypic change, transgene steady-state expression and alteration in flavonoid content in the resulting transgenic plants. These qualities were consistent within the transgenic plants obtained using any particular construct. Transgenic plants with decreased leaf naringin levels were obtained, particularly when the CHS-AS constructs were employed.

Citrus Transformation: Challenges and Prospects

Citrus is an important commodity worldwide and is produced in tropical and subtropical regions around the world. Annually, the total citrus fruit production is estimated to be more than 124.5 million tonnes worldwide, with China, Brazil, the United States, Mexico and India the main producers (FAO, 2011). Oranges, lemons, tangerines and grapefruits are among the most commonly grown citrus types and they are traded as fresh fruit, juice, or as concentrate. Growers, however, face important challenges for maintaining or improving yield: disease, drought, cold and soil salinity are some of the factors that can limit production and can have an important economic impact on growers. Traditional breeding methods have been used successfully over the years to improve citrus; however this is done with difficulty due to the slow growth and maturation of this crop, incompatibility, polyembryony, parthenocarpy, etc. Because traditional breeding takes such a long time the fast incorporation of desirable traits is not possible. In other instances, certain desirable traits are not present in cultivated citrus types. This has been made more evident in the battle against diseases. Diseases can appear in a region and within a few years spread and become limiting factors for production and have a major economical impact because of yield reduction and/or increased production costs. Therefore, genetic engineering via citrus transformation is an alternative method used to incorporate desirable traits into citrus genotypes.

A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from ‘Duncan’ grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and ‘Sun Chu Sha’ mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from ‘Nagami’ kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, ‘Nagami’ kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.