LIBRO-CONGRESO-CITRUS

engineering. We addressed this goal by blocking the expression of the endogenous β-carotene hydroxylase
gene (β-CHX), involved in the conversion of β-carotene into xanthophylls, using RNA interference technology.
Mature fruits of transgenic plants obtained showed changes in carotenoid content and composition in the
pulp, with an increase in β-carotene accompanied by a general decrease of xanthophylls. The putative
enhanced capacity of transgenic oranges for protection against oxidative stress and/or degenerative diseases
may be due to an additive/synergistic effect of increased β-carotene content in a background matrix rich in
vitamin C and other phytonutrients. This has been assessed by using Caenorhabditis elegans as experimental
animal model. Results of in vivo assays will be presented and discussed.
S05O06
RNA interference against the three Citrus tristeza virus genes encoding silencing suppressors
confers complete resistance to the virus in transgenic ‘Mexican’ lime plants
Soler N. 1 , Plomer M. 1 , Fagoaga C. 1 , Moreno P. 1 , Navarro L. 1 , Flores R. 2 , and Peña L. 1
1Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protección Vegetal y Biotecnología, Spain; and 2Instituto de Biología
Molecular y Celular de Plantas, CSIC-Universidad Politécnica de Valencia (IBMCP), Spain. soler_nurcal@gva.es
Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three
silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome
host antiviral defense. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has
been widely used for generating transgenic plant resistant against viruses. Considering the important role of p23,
p20 and p25 in CTV pathogenesis, we transformed ‘Mexican’ lime plants with an intron-hairpin vector carrying
full untranslatable versions of genes p25, p20, p23 and the 3’-UTR from the CTV strain T36, to attempt silencing
their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic
lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36,
either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived
siRNAs was necessary but not sufficient for CTV resistance. When immune transformants were challenged with
a dissimilar CTV strain the resistance was partially broken, stressing the importance of sequence identity in the
underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a
highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi.
S05O07
Further verification of the resistance of ‘C-05’ citron to Citrus Canker disease
Liu L.P., Li D.Z., Li J.H., Li N., and Deng Z.N.
National Center for Citrus Improvement (NCCI), Hunan Agricultural University, China. deng7009@163.com
In the previous screening of citrus genotypes, a Chinese citron (nominated citron ‘C-05’) was found resistant
to Canker disease (Xanthomonas axonopodis pv. citri). In order to verify its resistance, further tests were
performed. Another 4 local citron genoytpes from Yunan, including ‘Large’ citron, ‘Small’ citron or ‘Acid’
citron, ‘Java’ citron and ‘Indian Large Fruit’ citron, were inoculated in vivo to test their reaction to Xac. The
assay results indicated that all the 4 citron genotypes were less sensitive to the pathogen than sweet orange,
but all showed typical symptoms, while citron ‘C-05’ remained symptomless. The results provided further
evidence that citron ‘C-05’ is not non-host resistant. Growing leaves at different stages of ‘Bingtang’ sweet
orange (highly susceptible) and citron ‘C-05’ were inoculated by spraying with 10 8 CFU/mL Xac suspensions.
The leaves of ‘Bingtang’ sweet orange at 1/2 and 1/4 full size were highly susceptible, and leaves at other
stages were less susceptible; while in the citron, there were only few untypical disease spots on the leaves at
1/2 and 1/4 full size stages, while no reaction appeared in the other leaf stages. Inoculation with infiltration
of 10 10 , 10 8 , 10 6 , 10 4 , 10 2 and 10 CFU/mL bacterial solution all caused Canker disease on sweet orange leaves,
while citron ‘C-05’ showed hypersensitivity reaction. Bacterial growth on the leaf surfaces was observed
by electronic scanning microscope, and further by the use of a EGFP-labeled pathogen. The bacteria were
randomly distributed on the leaves, but on the citron leaf, bacterial growth was much less than that on sweet
orange leaf. Stomata observation revealed that the tested citrus genotypes were substantially similar. The
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S05