Infiltration-RNAseq: transcriptome profiling of Agrobacterium-mediated infiltration of transcription factors to discover gene function and expression networks in plants.

Donna M Bond,Gareth B Gillard,Robyn H Lee,Nick W Albert,Roger P Hellens,Richard C Macknight,Chris M Brown

doi:10.1186/s13007-016-0141-7

Abstract

BackgroundTranscription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed.ResultsAgroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis.ConclusionsInfiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method to identify TF-mediated transcriptional changes and TF target genes in M. truncatula and Nicotiana benthamiana. This included the identification of target genes of a TF not normally expressed in leaves, and targets of TFs from other plant species. Infiltration-RNAseq can be easily adapted to other plant species where agroinfiltration protocols have been optimised. The ability to identify downstream genes, including positive and negative transcriptional regulators, will result in a greater understanding of TF function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0141-7) contains supplementary material, which is available to authorized users.

Highlights

Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs
We propose a more simple experiment to understand TF-mediated gene expression changes that does not rely on the production of stable transformants—agroinfiltration of a TF followed by high-throughput RNA sequencing
Because infiltration of Agrobacterium into a leaf will cause a large number of transcriptional changes, such as activation of genes involved in the plant’s immune response (Additional file 7: Table S5), it was unclear if this method could be used to reliably identify endogenous genes that might be regulated by the transient overexpression of specific TFs

Summary

Introduction

Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. Transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. As some TFs display unique properties, the overexpression strategy has been effective in revealing TF function [1,2,3]. These methods rely on stable transformation of plant cells, but obtaining stable transformants of crop plants is a time-consuming and labourintensive process that is often inefficient. Only a low number of T-DNA copies integrate into the plant chromosomes, the non-integrated T-DNAs are transiently expressed for several days [5]

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Conclusion