A reporter for noninvasively monitoring gene expression and plant transformation

Abstract

Reporters have been widely used to visualize gene expression, protein localization, and other cellular activities, but the commonly used reporters require special equipment, expensive chemicals, or invasive treatments. Here, we construct a new reporter RUBY that converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. We show that RUBY can be used to noninvasively monitor gene expression in plants. Furthermore, we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis. The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

Keywords: Genetics; Plant biotechnology.

Fig. 1. Components required for betalain biosynthesis.

a Chemical reactions for converting tyrosine into betalain, which has a red color. Tyrosine is first oxidized by the P450 CYP76AD1 into l-3,4-dihydroxyphenylalanine (l-DOPA), which can be further converted into cyclo-DOPA by P450 CYP76AD1. In the presence of l-DOPA 4,5-dioxygenase (DODA), DOPA is oxidized and circularized into betalamic acid. Cyclo-DOPA condenses with betalamic acid, a non-enzymatic reaction, to produce betanidin. Glucoyslastion of betanidin generates the red color betalain. b A strategy for expressing the whole betalain biosynthetic pathway in a single cassette. The three betalain biosynthetic genes were fused into a single open reading frame, which can be expressed using a single promoter and terminator. Between the genes, sequences that encode 2A peptides were inserted. The 2A peptides undergo self-cleavage, thus releasing the individual enzymes for betalain biosynthesis. The betalain synthesis unit can be placed under the control of a promoter of interest. The terminator used here was the Arabidopsis HSP18.2 terminator. The open reading frame of 2A-linked betalain biosynthesis genes is named RUBY.

Fig. 2. RUBY serves as an effective reporter for gene expression and plant transformation.

a RUBY expression driven by the CaMV 35S promoter led to a red Arabidopsis plant (right) compared with the WT plant (left). b UBIQUITIN promoter was effective in driving RUBY expression throughout the plant (right). Non-transgenic WT was shown left. c The seed specific promoter At2S3 did not lead to RUBY expression in leaves and stems. The transgenic plant (right) and non-transgenic plant (left) were indistinguishable. The siliques of At2S3:RUBY were similar to those of WT, however, when the silique was opened, the seeds of At2S3:RUBY were clearly red. Moreover, it was obvious that transgenic and no-transgenic seeds were segregating in a silique from a T1 At2S3:RUBY plant. d YUC4:RUBY plants displayed patches of red at the tip of leaves and apical region of a gynoecium. e DR5:RUBY was expressed in rice calli. The red color can be used to distinguish transgenic (red) and non-transgenic calli (white). DR5:eGFP has been used in rice calli f, but it was much more difficult to distinguish transgenic from non-transgenic using DR5:eGFP compared with RUBY. g Roots of DR5:RUBY and DR5eGFP rice plants, which had similar patterns. The three DR5:eGFP pictures were generated with the same root: bright field (left), 488 nm fluorescence field (middle), and the merged (right). h Activation of DR5 promoter in DR5:RUBY leaves was easy to observe whereas DR5:eGFP was much more difficult to detect: bright field (left), 488 nm fluorescence field (middle), and the merged (right).