Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system

Abstract

SummaryWe used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T 0 adult plants (1982 lines), T 1 seed (2684 lines) and T 1 seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.

Brassinosteroids Regulate Grain Filling in Rice

“…Imaging (Figures 1A to 1F) and RT-PCR analysis ( Figure 1K) of green fluorescent protein (GFP) expression in rice plants carrying a two-component construct containing pAS, the yeast transcriptional activator HAP1, five tandem repeats of the yeast upstream activating sequence recognized by HAP1 (HAP1 UAS ), and GFP (pAS:HAP1:UAS HAP1 :GFP [aHAP]; see Supplemental Figure 1 online) showed that pAS directs expression to many or all of the cells in the stems, leaves, and roots of rice but not to the flowers other than weakly in the filaments of the anthers or to the seed other than weakly in the epidermis of the seed coat. In these plants, HAP1 operated in cis to activate the HAP1 UAS repeats and drive amplified expression of GFP (Zhang and Guarente, 1994;Johnson et al, 2005). Imaging of plants containing a pAS: GFP direct fusion showed a similar, but weaker, expression pattern.…”

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants

“…L4, Gateway recombination site attL4; R1, attR1; L1, attL1; L2, attL2; R2, attR2; L3, attL3; B4, attB4; B1, attB1; B2, attB2; B3, attB3; XVE, chimeric transcription factor containing the DNAbinding domain of LexA, the transcriptional activation domain of VP16, and the regulatory domain of the human estrogen receptor; e9T, rbcS E9 poly(A) addition sequence; nosT, nopaline synthase poly(A) addition sequence; pLexA, eight copies of the LexA operator sequence together with a minimal cauliflower mosaic virus 35S promoter. Odell et al, 1985) Yes (data not shown) p1R4-p6xUAS:XVE Upstream activation sequence containing several GAL4-binding elements and minimal 35S (Brand and Perrimon, 1993;Johnson et al, 2005) Yes (Vatén et al, 2011) p1R4-pAHP6:XVE Protoxylem + protoxylem-pericycle, organ primordia in shoot apical meristem/ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6)/AT1G8010 (Mähö nen et al, 2006;Bartrina et al, 2011;Besnard et al, 2014) Yes (Mähö nen et al, 2014) p1R4-pANT:XVE Developing ovules, primordia of all lateral shoot organs/ AINTEGUMENTA/AT4G37750 (Elliott et al, 1996) Yes (data not shown) p1R4-pAPL:XVE Phloem (developing sieve elements and companion cells)/ALTERED PHLOEM DEVELOPMENT (APL)/AT1G79430 (Bonke et al, 2003) Yes (Bishopp et al, 2011b;Vatén et al, 2011) p1R4-pAtSUC2:XVE Phloem companion cells/ARABIDOPSIS THALIANA SUCROSE-PROTON SYMPORTER2/AT1G22710 (Truernit and Sauer, 1995) Yes (data not shown) p1R4-pCALS3:XVE Vascular bundle, root stem cell niche/CALLOSE SYNTHASE3 (CALS3)/ AT5G13000 (Vatén et al, 2011) Yes (Vatén et al, 2011) p1R4-pCCS52A1:XVE Root elongation zone, lateral roots/CELL CYCLE SWITCH PROTEIN52 A1 (CCS52A1)/AT4G22910 (Vanstraelen et al, 2009) Yes (data not shown) p1R4-pCO2:XVE Cortex/CORTEX2 (CO2)/AT1G62500 (Heidstra et al, 2004) Yes (Dhonukshe et al, 2010; this article) p1R4-pCYP79B3:XVE Meristem quiescent center and the surrounding stem cells/ CYTOCHROME P450, FAMILY 79, SUBFAMILY B3 (CYP79B3)/ AT2G22330 (Ljung et al, 2005) Yes (data not shown) p1R4-pG1090:XVE Strong ubiquitous expression/G10-90 (artificial promoter containing repeats of G-box; Ishige et al, 1999) Yes ( Yes (Bishopp et al, 2011a;Vatén et al, 2011;Ursache et al, 2014; this article) p1R4-pWOX5:XVE Root meristem quiescent ce...…”

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures

“…The reporter gene will be activated if the construct is integrated into a position adjacent to an enhancer (or a promoter). A large number of enhancer/ promoter trapping lines were developed in several plant species, including Arabidopsis (Sundaresan et al, 1995;Campisi et al, 1999), rice (Oryza sativa) (Wu et al, 2003;Yang et al, 2004;Johnson et al, 2005), and poplar (Populus trichocarpa) (Groover et al, 2004). Unfortunately, the enhancer trapping approach has a major drawback.…”