GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Abstract

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

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).…”

“…'Pêra' and 'Valência' sweet orange cultivars were selected as the genetic material for the transformation experiments. Seed extraction, explant preparation, transformation, and plant acclimation procedures were carried out according to Miyata 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).…”

“…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).…”

Transformação genética de laranjas 'Pera' e 'Natal' (Citrus sinensis L. Osbeck) com o gene atacina A (attA), dirigido por promotores preferencialmente expressos no floema, para resistência a Candidatus Liberibacter spp.

“…Desse modo, muitos estudos estão sendo realizados, com resultados promissores, utilizando promotores de expressão preferencial no floema, como AtSuc2 (transportador de sacarose), AtPP2 (proteína de floema 2) clonados de Arabidopsis thaliana, CsPP2 (proteína de floema 2) clonado de Citrus sinensis (MIYATA et al, 2012;TAVANO, 2013) (DUTT et al, 2012;BENYON et al, 2013).…”