Development of transactivator system for rice and its application for studies of floral development

Abstract

The development of a system for the discovery of rice genes and their function is a high priority. Two different B-glucuronidase reporter gene cassettes (GUS and GUSPlus) and two GAL4/VP16 transactivator cassettes (with and without the catalase- 1 intron upstream of the GAL4/VP16) as well as their relative position (1.6 kb and 7 kb relative distances between the upstream activating sequence (UASGAL4) and the CaMV35S promoter driving a hygromycin gene) within T-DNA were tested for their performance in the Transcriptional Activator-Facilitated Enhancer trap (TAFET) constructs to induce cell- or tissue-specific expression patterns in rice. A total of 1 0 TAFET constructs, including two negative controls, were developed and introduced into the rice genome of varieties Millin and Nipponbare using Agrobacterium-mediated transformation and about 1,000 rice TAFET lines were produced and investigated. The GAL4/VP16 TAFET system was able to reveal varying levels of tissue specific expression patterns in rice in the vegetative and generative tissues, and the expression patterns were proven to be due to the activity of the GAL4/VP16 transcriptional activator. Most of the TAFET lines examined showed reporter gene expression in many tissues. Patterns shown often in the vascular bundle, pericycle layer of cells in the root suggest that the CaMV35S enhancers affected the expression patterns in numbers of lines. However, the observed patterns are likely to be primarily due to a complex interaction between rice genomic endogenous enhancers and elements within T -DNAs. Most patterns observed in TAFET lines (91.7%) were inherited by the next generations (T1 and T2) and segregated in Mendelian fashion. T-DNA inserts segregated among progeny plants in agreement with expected ratios. About 49% of VI lines contained a single T-DNA insertion and the average number of T-DNAs in the whole TAFET population was 2.0. The crossing experiments between selected GUS-TAFET lines and selected Enhancer Green Fluorescence protein (EGFP) target lines proved that the GAL4/VP16 TAFET system is capable of activating a gene (a reporter gene) adjacent to UAS. The most likely mechanism of activation is through the GAL4 DNA binding domain recognising the UASGAL4 , and the VP16 activating domain promoting transcription of a gene linked to the UASGAL4 . Expression patterns of EGFP reporter gene in F1 plants were in general consistent with GUSPlus patterns observed in parental GUS-TAFET lines. Attempts to generate phenotypic changes in floral tissues by induced ectopic expression of the ZAG 1 target gene from maize did not fully succeed, as the changes were only limited to some spikelets. The positive control in these experiments, 35S promoter driven over-expression of ZAG1, produced limited phenotypic consequences (increased sterility in some plants), suggesting that the inability of the TAFET system to induce clearly observable changes in floral development were due to poor expression of the ZAG 1 gene. A number of TAFET lines showed phenotypic changes that segregated in T1 and T 2 generations. An attempt to clone a gene responsible for a dramatic change in floral development in line pSKC66.1-8e failed due to the lack of a link between T-DNA insertion and mutant phenotypes. Tos 17 retrotransposon mobility during tissue culture was determined to be the most likely cause of floral development change observed in this line. vii In general, good performance of the GAL4/VP16-based TAFET system was determined both through observations of TAFET lines in several generations and through sexual crossing with TARGET EGFP lines. The implication of this research is that the system can be used to direct the expression of any gene under study (a target) in specific tissue types to generate ectopic expressions and possibly also Gain-of Function (GoF) phenotypes. The TAFET system therefore can be applied in rice functional genomics studies and could possibly contribute to the generation of a novel germplasm with useful traits for agriculture.