Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.

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Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs. Since the production of transgenic citrus lines and their evaluation would take years, a herbaceous model plant, Nicotiana benthamiana, was used to test hairpin constructs. The expression of self-complementary hairpin RNA fragments from the coat protein (cp) and 54K genes of the Argentine CPsV 90-1-1 isolate conferred resistance on N. benthamiana plants, indicating that these constructs are good candidates for the transformation of citrus plants. The degree of resistance obtained varied depending on the viral sequence chosen. The analysis of the levels of small interfering RNA accumulation and viral RNAs indicated that the construct derived from cp gene was better at inducing PTGS than that originating from the 54K gene. The dependence of PTGS induction on the degree of identity between the target and the inducer transgene sequences was tested using sequences derived from CPV4, a more distant isolate of CPsV, as PTGS targets. Efficient silencing induction was also obtained to this isolate through the expression of the cp-derived hairpin. This is the first report of transgenic-resistant plants within the context of this serious citrus disease.

Citrus psorosis is a widespread serious disease of citrus caused by Citrus psorosis virus (CPsV). In Argentina and Uruguay, this disease is spread by an unknown vector and there is no natural resistance or tolerance to the disease. There are two types of psorosis, described according to the symptoms observed in citrus trees, psorosis A (PsA) and psorosis B (PsB). PsA protects against the severe effects of the more aggressive type PsB. We have applied pathogen‐derived resistance to create a defence mechanism against this virus disease. Sweet orange transgenic lines were obtained containing three different genes of CPsV (54k, 48k and 24k genes) taken from a PsA isolate (CPV‐4). Fourteen lines were selected containing 1, 2 or 3 copies of the transgenes and evaluated for their acquired resistance against PsA (CPV 4 from USA) and PsB (CPsV 189‐34 from Argentina) isolates. These lines were susceptible to both isolates when graft‐infected, although one of the lines carrying the cp gene (CP‐96 line) containing two copies of the transgene and expressing a low level of the coat protein showed a delay in symptom expression when inoculated with the PsB isolate.

Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions.

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Actualmente, no se conocen especies cítricas ni especies relacionas utilizadas como pie en la citricultura que sean resistentes o tolerantes al virus de la psorosis de los cítricos. Por sus características epidémicas, el control de la enfermedad y su erradicación son difíciles de llevar a cabo. La resistencia deriva del patógeno (PDR) se ha aplicado con éxito para el control de diversas enfermedades producidas por agentes virales. En muchos casos la expresión de la proteína de la cápside viral u otras secuencias virales, ha generado resistencia o tolerancia al virus sin producir efectos secundarios. Por lo tanto, uno de los objetivos generales planteado en la presente tesis fue aplicar la estrategia de resistencia derivada del patógeno al virus de la psorosis de los cítricos en búsqueda de resistencia a la enfermedad. Dado que en los virus la información genética se encuentra muy condensada, es frecuente encontrar que la mayoría de sus genes están involucrados en varias de las funciones necesarias para completar el ciclo viral. CPsV posee 4 ORFs determinados a partir del análisis de su genoma, de los cuales, solamente se ha asignado y confirmado su función a la proteína 48K (proteína de cubierta viral) y determinado por homología de secuencia la función de replicasa viral en la proteína 280K. Por lo tanto, además de la función estructural de la proteína 48K es muy probable que participe de otras funciones vitales para la replicación e infección del virus. El análisis de la secuencia nucleotídica de la proteína 280K del RNA1 (RNA polimerasa) y de la proteína 54K parece indicar que podrían ejercer alguna función en el núcleo ya que poseen señales de localización nuclear. En general, el análisis de la localización subcelular de las proteínas virales a posibilitado dilucidar la función de las mismas durante el ciclo vital, por lo cual, se estableció como segundo objetivo general de la tesis estudiar la expresión y/o localización intracelular de las proteínas virales de CPsV. Para lograr dichos objetivos, durante el trabajo de tesis se abordaron diferentes objetivos particulares que se desarrollaron en los capítulos que la conforman: Capítulo 1. Obtención de plásmidos recombinantes utilizados durante el desarrollo de la tesis Capítulo 2. Desarrollo de herramientas y metodologías para la detección de las proteínas 24K, 48K y 54K de CPsV Capítulo 3. Búsqueda de resistencia derivada del patógeno mediante la obtención de cítricos transgénicos conteniendo ORFs de CPsV. Capítulo 4. Estudio de la expresión de la proteínas 24K, 48K y 54K y la localización sub-celular de la proteína de cubierta viral de CPsV. Capítulo 5. Detección de CPsV aplicando el TAS-ELISA-HRP