How to drive phloem gene expression? A case study with preferentially expressed citrus gene promoters

Como regular a expressão de genes no floema? Um estudo de caso com promotores de genes de citros preferencialmente expressos

Abstract

New approaches for developing disease-resistant genetically modified organisms have included specific targets for gene expression to enhance the chances for pathogen control. Gene expression driven by phloem-derived Citrus sinensis gene promoters could be evaluated and compared with the expression induced by a strong constitutive promoter in the same tissue, leading to the production of transgenic sweet oranges potentially more resistant to diseases caused by phloem-limited bacteria. ‘Carrizo’ citrange [ (Poncirus trifoliataL.) Raf. x Citrus sinensis (L.) Osbeck] was transformed, via Agrobacterium tumefaciens, with the binary vector pCAMBIA2301 bearing the uidA gene (ß-glucuronidase) driven by the CaMV35S constitutive promoter (CaMV35S::uidA) or by the CsPP2.B1 (CsPP2.B1::uidA) or by the CsVTE2 (CsVTE2::uidA) citrus promoters. In vitro regenerated shoots were grafted onto ‘Rangpur’ lime (C. limonia Osbeck). The genetic transformation was confirmed by Southern blot analyses. uidA gene expression was evaluated by RT-qPCR, and gene histolocalization controlled by these three promoters was accessed by X-GLUC treated stem sections. uidA gene expression exhibited by tissue-specific promoters was overall lower than from the constitutive promoter CaMV35; however, constructs driven by tissue-specific promoters may lead to expression in restricted tissues. CsPP2.B1 and CsVTE2 promoters can be considered adequate for the utilization in gene constructs aiming disease resistance.

Index terms
Candidatus Liberibacter spp.; disease resistance; genetic transformation; GUS; histolocalization