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

Abstract

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.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp. citri

“…Citrus cultivars have been genetically modified aiming mainly disease resistance, such as those caused by Phytophthora sp. (Fagoaga et al 2001), Phoma tracheiphila and Botrytis cinerea (Gentile et al 2007;Distefano et al 2008), Citrus tristeza virus (Domínguez et al 2000;Febres et al 2003Febres et al , 2008, and Citrus psorosis virus (Zanek et al 2008). Several approaches have been used to engineer plants for disease resistance, such as direct interference with pathogenicity or inhibition of pathogen physiology (including constitutive expression of antimicrobial factors), pathogen-induced expression of one or more genes, and regulation of the natural induced host defenses (including altering recognitions of the pathogen such as R-genes and downstream regulatory pathways such as systemic acquired resistance (SAR); Collinge et al 2008).…”

Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

“…Genetic transformation provides the means for adding a single agronomic trait in perennial plant cultivars without altering their phenotype (Deng and Duan 2006). Until now, various genes have been introduced into citrus species and its relatives to improve agronomical traits, including enhancing abiotic stress tolerance (Cervera et al 2000;Fagoaga et al 2007) and disease resistance (Ananthakrishnan et al 2007;GonzalezRamos et al 2005;Omar et al 2007;Zanek et al 2008), shortening the juvenile phase (Duan et al 2007a;Endo et al 2005;Peña et al 2001), rootstock (Gentile et al 2004), and fruit improvement (Costa et al 2002;Guo et al 2005;Koltunow et al 2000;Li et al 2002;Wong et al 2001). Among these, fruit improvement is of particular importance as it can be very attractive for consumers.…”

Transformação genética de laranja doce (Citrus sinensis L. Osbeck) com o gene D4E1 dirigido pelos promotores CaMV35S ou AtPP2

“…Neste trabalho, cada planta transgênica apresentou diferentes padrões de integração e diferentes números de inserções do transgene no genoma, variando de uma a três cópias. Este valor está dentro do observado por outros autores em plantas de citros transgênicas de uma a quatro inserções (DUTT et al, 2012;REYES et al, 2011;ZANEK et al, 2008). Futuramente, estas plantas serão multiplicadas e avaliadas quanto à resistência ao cancro cítrico e ao HLB.…”