Gene Silencing of viral 24K- gene induces uncontrolled infection of Citrus psorosis virus (CPsV) but not of the unrelated Citrus tristeza virus (CTV)

CARINA REYES; AGUSTINA DE FRANCESCO; NORMA COSTA; MARIA LAURA GARCIA

Mpumalanga

Conferencia; IXX Conference of the IOCV; 2013

International Orgnization of Citrus Virologists (IOCV)

The Ophoivirus Citrus psorosis virus (CPsV) is the causal agent of a serious disease affecting citrus trees in many countries. Post-transcriptional gene silencing (PTGS), can be induced by transgenic expression of pathogen-derived sequences encoding hairpin RNAs. Using this strategy is possible to knock down gene expression in order to study gene functions. Citrus sinensis plants were transformed with a hairpin construct derived from 24k gene (ihp24K) from the Argentine CPsV 90-1-1 isolate. Several independent ihp24k transgenic lines were generated and after regeneration, two of these lines (6117 and 6119) were propagated in the greenhouse and challenged by graft-inoculation with CPsV 90-1-1. Symptoms observation and molecular analyses (RT-PCR and TAS-ELISA) were performed. Both challenged lines ihp24k were highly susceptible to the virus when assayed under greenhouse conditions (18-24 ºC), showing more severe symptoms than non transgenic control, and presented higher viral titers. The symptoms were manifested in successive flushes including flecking, spots and shock reaction reflecting an uncontrolled infection in the 24K silenced plants. To gain insight into the specificity of the hypersusceptibility triggered in the 24K silenced plants, a second assay was performed challenging 12 propagations of both ihp24K lines and non transgenic control plants either with the unrelated Closterovirus Citrus Tristeza Virus (CTV) or with CPsV 90-1-1. Symptoms and ELISA evaluation reflects the expected severe symptoms in the CPsV inoculated transgenic plants compared to the non transgenic control plants but first evaluation of the CTV inoculated transgenic plants did not show the same clear hyper-symptomatic behavior. Further evaluations of the inoculated plants along time should be conducted but these results suggest and specific mechanism triggered in the 24K silenced plants leading to an unregulated infection. Studies focused to the understanding of the molecular mechanisms behind uncontrolled infection expressed by these lines should be also running in order to unravel this putative 24K regulation function of the viral cycle.