Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants

Abstract

'Mexican' lime (Citrus aurantifolia Swingle) was transformed with constructs that contained chimeric promoter-gus gene fusions of phloem-specific rolC promoter of Agrobacterium rhizogenes, Arabidopsis thaliana sucrose-H(+) symporter (AtSUC2) gene promoter of Arabidopsis thaliana, rice tungro bacilliform virus (RTBV) promoter and sucrose synthase l (RSs1) gene promoter of Oryza sativa (rice). Histochemical β-glucuronidase (GUS) analysis revealed vascular-specific expression of the GUS protein in citrus. The RTBV promoter was the most efficient promoter in this study while the RSs1 promoter could drive low levels of gus gene expression in citrus. These results were further validated by reverse transcription real-time polymerase chain reaction and northern blotting. Southern blot analysis confirmed stable transgene integration, which ranged from a single insertion to four copies per genome. The use of phloem-specific promoters in citrus will allow targeted transgene expression of antibacterial constructs designed to battle huanglongbing disease (HLB or citrus greening disease), associated with a phloem-limited Gram-negative bacterium.

Genetic transformation of sweet oranges with the D4E1 gene driven by the AtPP2 promoter

“…Miyata et al (2012) demonstrated specific GUS expression driven by the AtPP2 promoter in phloem tissue of transgenic sweet orange. However, uidA gene expression driven by this type of promoter, quantified by qPCR, is relatively lower when compared to genes driven by constitutive promoters (Dutt et al, 2012;Benyon et al, 2013).…”

“…Noticeably, no event showed gene silencing according to the qPCR analysis results because all transformation events presented gene expression. Gene silencing is typically detected in plants with multiple copies integrated at one or several loci; however, gene silencing was not reported in citrus plants with up to four transgene copies (Dutt et al, 2012;Benyon et al, 2013).…”

“…Several promoters have already been identified for tissue-specific expression, including those that preferentially induce expression in the phloem, such as Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) (Singer et al, 2011), Arabidopsis thaliana phloem protein 2 (AtPP2), C. sinensis phloem protein 2 (CsPP2), A. thaliana sucrose transporter 2 (AtSUC2) (Miyata et al, 2012), and rice sucrose synthase-1 (RSs-1) (Wang et al, 2005;Dutt et al, 2012). In addition, promoters not originated from plants, such as those from phloem-limited virus genes, including the Coconut foliar decay virus (CFDV) (Hehn & Rohde, 1998) and the Rice tungro bacilliform virus (RTBV), have also been used as promoters with strong expression in citrus plants (Dutt et al, 2012).…”

“…In addition, promoters not originated from plants, such as those from phloem-limited virus genes, including the Coconut foliar decay virus (CFDV) (Hehn & Rohde, 1998) and the Rice tungro bacilliform virus (RTBV), have also been used as promoters with strong expression in citrus plants (Dutt et al, 2012). The preferential expression of the β-glucuronidase enzyme in phloem was shown through uidA gene expression in transgenic citrus driven by the AtPP2, CsPP2, AtSUC2, RSs-1, and RTVB promoters (Dutt et al, 2012;Miyata et al, 2012).…”

“…The expression of an antimicrobial peptide in the citrus plant phloem, particularly in young leaves, can aid in controlling HLB because this disease is associated with bacteria restricted to phloem cells, which are primarily spread by psyllids while feeding on young leaf phloem (Dutt et al, 2012). Another advantage of preferential expression in phloem is that it decreases the expression of the transgene in other tissues (Zhao et al, 2004).…”