Author: Customer
Company: ARO

ABSTRACT
Citrus is the most extensively grown fruit tree crop in the world and a major source of income and employment. Citrus tristeza virus (CTV) (genus closterovirus family Closteroviridae) is involved in a range of serious citrus diseases that limits production by causing decline of trees grafted on sour orange rootstocks and by stem-pitting that debilitates trees and reduces fruit quality regardless of rootstock. The aphid transmissible CTV virions are long, flexuous particles and contain the largest and most complex single stranded RNA genome among plant viruses. The CTV host range is mostly restricted to Rutaceous plants. Genetic improvement of citrus rootstocks to overcome their sensitivity to CTV remains an important objective for citrus industry. Recent developments in genetic engineering allows the expression of different viral genes in plants and resulted in transgenic plants with considerable levels of resistance to homologous virus infections. Citrus plants are recalcitrant to Agrobacterium-mediated genetic engineering and give only low in vitro regeneration efficiency. Therefore the task of transgenic improvement remains a most challenging objective. The effects of different conditions on the efficiency of the regeneration process and the ability to obtain transgenic citrus rootstocks were examined. The results of Agrobacterium-mediated transformation in vitro are reported for the five most economically important citrus rootstocks in etiolated epicotyl
stem segments of Troyer citrange (Citrus sinensis x Poncirus trifoliata), Sour orange (C. aurantium), Gou Thou (Chinese sour orange), Alemow (C. macrophylla), and Volkamer lemon (C. limon). Agrobacterium-mediated transformation was attempted with several CTV derived sequences including CTV p23 with 3’ UTR (p23U, ORF 11), p61 (ORF 5) and the hairpin structure of p23U (p23UI). In addition to citrus transformation, we prepared transgenic Nicotiana benthamiana plants expressing the corresponding CTV sequences used for citrus transformation and used this system for the rapid analysis of construct and cloning efficiency. The availability of the Grapevine Virus A (GVA) based viral vector, infectious on N. benthamiana allowed the assessment of the transgenic N. benthamiana plants for PTGS-based resistance against a recombinant GVA vector containing the corresponding homologous CTV sequences. In total the citrus rootstocks transformation efforts resulted in more than 300 transgenic citrus rootstocks and 435 transgenic N. benthamiana plants that have tested positive for the presence of CTV specific sequences as indicated by a battery of tests including GUS, PCR and Southern hybridization with CTV- p61 and p23U gene specific DIG-labeled RNA probes. Hybridization of RNA extracts from either of these classes of transgenic plants with riboprobes specific to CTV-p61 and plus- and minus-strand p23U molecules demonstrated that most of the transgenes contained the expected size transcription products as RNA and as dsRNA molecules. To test the effectiveness of CTV-p61, -p23U and -p23UI constructs to confer resistance when present in transgenic citrus and N benthamiana hosts, the plants were challenged with two types of virus inocula: (I) the authentic CTV and (II) an infectious GVA, harboring CTV-p23U and -p61. All tested transgenic p23UI N. benthamiana plants challenge-inoculated with the GVA vector harboring the p23U were highly resistant to the chimeric virus. In contrast, when experiments were conducted with transgenic citrus plants harboring similar cDNA constructs, none of the challenged plants were found to show durable resistance against the authentic CTV inoculum introduced by graft inoculation. We categorized the challenged transgenic citrus plants by their symptom development; those that showed a) severe symptoms like non transgenic plants b) short and/or long delay on symptom onset, c) recovered and d) non-visible symptoms. None of the CTV-derived sequences used in this study resulted in durable resistance. Both, transgenic and non transgenic citrus plants infected by CTV accumulated detectable levels of viral-specific siRNAs from various parts of the genome indicated that, irrespective of the presence or absence of viral-derived transgenes, CTV is the target of a PTGS in nature. Sequence analysis of CTV genome from authentic and recovered strains revealed differences in nucleotide composition and these findings will be useful in future studies aimed to locate the
“Seedling Yellows” pathogenicity determinant. The possible causes for the failure of the transgenes to confer durable resistance to CTV in transgenic citrus plants are discussed.