Differential resistance to Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA derived from the coat protein and 54K protein genes.

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.

Detection of Citrus psorosis virus in the northwestern citrus production area of Argentina by using an improved TAS-ELISA.

Citrus Psorosis in Argentina is a serious disease. Citrus is produced in two regions located in the northeast (NE) and northwest (NW) area of the country. These two areas have different climates and soil types, and therefore different citrus species and varieties are cultivated. In the NE region, Psorosis is epidemic, and in the NW region, the disease was described on several occasions since 1938, but it is not observed commonly in the orchards. Recently, trees with symptoms of Psorosis were observed in the Tucumán and Salta Provinces located in the NW region. Epidemiological studies in Argentina and Texas suggested that the disease is spread naturally by an unknown vector. The causal agent of the disease is the Citrus psorosis virus (CPsV), which can be detected by TAS-ELISA, RT-PCR and indicator plants. A new more rapid TAS-ELISA-HRP (horseradish peroxidase) is described which is more reliable, faster and more sensitive than the currently used for this virus, the TAS-ELISA-AP (alkaline phosphatase). Psorosis was detected by this improved method in few trees in the orchards of the Tucumán Province, in the NW citrus region, although natural spread does not seem to occur.

Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease.

Citrus canker provoked by Xanthomonas axonopodis pv. citri is a bacterial disease causing severe losses in all citrus-producing areas around the world. Xanthomonas infection is considered as an endemic disease in Northeast and Northwest Argentina, affecting as much as 10% of commercial citrus plantations. There is not known natural resistance neither in orange varieties nor in rootstocks used for grafting of commercial cultivars. To introduce resistance to this disease, plants of Pineapple sweet orange were transformed with a genetic construct allowing constitutive accumulation of dermaseptin. In comparison with non-transformed plants, transgenic plants showed symptom reduction levels of up to 50% in in planta assays performed under controlled conditions.

Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo.

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.

Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus.

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.