HAIRPIN RNA EXPRESSION FROM CITRUS PSOROSIS VIRUS 24K-GENE ENHANCES SYMPTOMS OF PSOROSIS IN CITRUS SINENSIS

REYES CA; DE FRANCESCO A; COSTA N; PLATA MI; GARCIA ML

Campinas, Brazil

Conferencia; XVIII Conference of the International Organization of Citrus Virologists (IOCV); 2010

IOCV

Citrus psorosis virus (CPsV) is the causal agent of a serious disease affecting citrus trees in many countries. This ophiovirus is tripartite and its genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of RNA degradation involved in plant protection against virus, can be induced by transgenic expression of pathogen-derived sequences encoding hairpin RNAs. Using this strategy, Citrus sinensis plants were transformed with three hairpin constructs obtained from different genome regions of the Argentine CPsV 90-1-1 isolate; the coat protein (cp) from RNA 3, the 54k gene from RNA 2 and the 24k gene from RNA 1, which also encodes the RNA polymerase. Eight ihpCP, seven ihp24k and nine ihp54k lines were generated and after regeneration, some of the lines were propagated in the greenhouse and challenged by graft-inoculation with the Argentine isolate. Acquired resistance was evaluated by symptoms observation and molecular analyses (RT-PCR and TAS-ELISA). Different levels of resistance were obtained: particularly lines ihp24k (6117 and 6119) were highly susceptible to the virus when assayed under greenhouse conditions (18-24 ºC). After challenge, they showed 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, while in the non transgenic controls symptoms normally decrease after first and second flushes. Besides, virus is more homogeneous distributed in the ihp24K plants than in the controls. Analysis by Northern blots to detect small interfering RNAs (siRNAs) rendered by the PTGS machinery and derived from the ihp24K transgene, were performed. Very low levels of specific siRNAs were detected in the non-inoculated ihp24K plants and higher in the infected samples. Further studies focused to the understanding of the molecular mechanisms behind hyper susceptibility expressed by these lines should be conducted. They would help us to gain more insight into viral cycle and plant – virus interaction.