GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters.
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.
Departamento de Produção Vegetal, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, 13418-900, Brazil.
Luzia Yuriko
LY
Miyata
Ricardo
R
Harakava
Liliane Cristina Libório
LC
Stipp
Beatriz Madalena Januzzi
BM
Mendes
Beatriz
B
Appezzato-da-Glória
Francisco
F
de Assis Alves Mourão Filho
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics; D032084:Citrus sinensis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics; D009928:Organ Specificity; D052585:Phloem / Q000235:genetics* / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D037121:Plant Lectins / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D018547:Plant Stems / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012038:Regeneration; D012230:Rhizobiaceae / Q000502:physiology; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D026901:Membrane Transport Proteins; C552547:PP2-A1 protein, Arabidopsis; D037121:Plant Lectins; D010940:Plant Proteins; C078729:sucrose transport protein, plant; D005966:Glucuronidase