|Shatters, Robert - Bob|
Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2013
Publication Date: 6/1/2013
Citation: Benyon, L.S., Stover, E., Bowman, K.D., Niedz, R.P., Shatters, R.G., Zale, J.,M., Belknap, W.R. 2013. GUS expression driven by constitutive and vascular specific promoters in citrus hybrid US-802. In Vitro Cellular and Developmental Biology Plants. 49:255-265. Interpretive Summary: Biotechnology is being used to develop resistance in citrus to the disease huanglongbing (HLB) also known as citrus greening. The promoter used is critical in making genetically engineered plants since it determines where genes are expressed in the plant, and how strongly the gene is turned on. This study compares the characteristics of five promoters driving a marker gene in citrus hybrid rootstock US-802. Because the bacterium associated with HLB infects only the citrus phloem tissue (plant plumbing that carries sugars and other important plant chemicals throughout the tree),it may be advantageous to limit transgene expression to this region and reduce expression in the fruit. We studied three promoters with phloem specificity when transformed and expressed in other plants, and two that generally express genes throughout the plant (known as constitutive expression). Marker gene activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem promoters largely showed the expected phloem-specific staining. Assessment of messenger RNA (first product when genes are expressed which then directs formation of the marker protein in our system) revealed a wide range of abundance from transgenics among all five promoters. Levels of the marker messenger RNA and marker protein activity did not correlate indicating possible effects of promoters on levels of marker protein produced per unit of messenger RNA. This information will help us in developing disease-resistant citrus.
Technical Abstract: Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus, and a critical component of the transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters driving the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants, the tandem repeat promoter of cauliflower mosaic virus 35S (2X35S) gene and bul409S a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the gram negative bacterium associated with HLB, infects only the citrus phloem tissue, it may be advantageous to limit transgene expression to this tissue and reduce expression in the fruit. Thus we chose three promoters that demonstrate phloem specificity when transformed and expressed in other plants, WDV from wheat dwarf geminivirus, AtSUC2 the sucrose-H+ symporter gene promoter from Arabidopsis, and CsSUS the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem promoters largely showed the expected tissue-specific staining. There were individual transformants with promoters CsSUS and WDV whose expression appeared leaky with some laminar tissue staining. Relative quantification of RT-qPCR data revealed a wide range of mRNA abundance from transgenics among all five promoters. Fluorometry also revealed that translation of GUS under each promoter varied, but the two data sets did not correlate indicating possible post-transcriptional modifications.