Inoculation insensitive promoters for cell type enriched gene expression in legume roots and nodules.

Srdjan Gavrilovic,Zhe Yan,Jens Stougaard,Anna M Jurkiewicz,Katharina Markmann

doi:10.1186/s13007-016-0105-y

Abstract

BackgroundEstablishment and maintenance of mutualistic plant–microbial interactions in the rhizosphere and within plant roots involve several root cell types. The processes of host–microbe recognition and infection require complex signal exchange and activation of downstream responses. These molecular events coordinate host responses across root cell layers during microbe invasion, ultimately triggering changes of root cell fates. The progression of legume root interactions with rhizobial bacteria has been addressed in numerous studies. However, tools to globally resolve the succession of molecular events in the host root at the cell type level have been lacking. To this end, we aimed to identify promoters exhibiting cell type enriched expression in roots of the model legume Lotus japonicus, as no comprehensive set of such promoters usable in legume roots is available to date.ResultsHere, we use promoter:GUS fusions to characterize promoters stemming from Arabidopsis, tomato (Lycopersicon esculentum) or L.japonicus with respect to their expression in major cell types of the L.japonicus root differentiation zone, which shows molecular and morphological responses to symbiotic bacteria and fungi. Out of 24 tested promoters, 11 showed cell type enriched activity in L.japonicus roots. Covered cell types or cell type combinations are epidermis (1), epidermis and cortex (2), cortex (1), endodermis and pericycle (2), pericycle and phloem (4), or xylem (1). Activity of these promoters in the respective cell types was stable during early stages of infection of transgenic roots with the rhizobial symbiont of L.japonicus, Mesorhizobium loti. For a subset of five promoters, expression stability was further demonstrated in whole plant transgenics as well as in active nodules.Conclusions11 promoters from Arabidopsis (10) or tomato (1) with enriched activity in major L.japonicus root and nodule cell types have been identified. Root expression patterns are independent of infection with rhizobial bacteria, providing a stable read-out in the root section responsive to symbiotic bacteria. Promoters are available as cloning vectors. We expect these tools to help provide a new dimension to our understanding of signaling circuits and transcript dynamics in symbiotic interactions of legumes with microbial symbionts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0105-y) contains supplementary material, which is available to authorized users.

Highlights

Establishment and maintenance of mutualistic plant–microbial interactions in the rhizosphere and within plant roots involve several root cell types
Nodule expression patterns To evaluate the suitability of the promoters for directed expression studies at later stages of nodulation symbiosis in L. japonicus, we investigated the activity patterns of pLeExt1:GUS, pAtCortex:GUS, pAtE29:GUS, pAtS13:GUS, and pAtS18:GUS in nodule cell types (Fig. 3a–c) using selected transgenic lines (Table 3)
Our results suggest that while the tested promoters may show broader expression ranges in L. japonicus compared to the endogenous Arabidopsis, higher detection sensitivity using the GUS reporter system may reveal low levels of expression present in other than the dominant cell type(s)

Summary

Introduction

Establishment and maintenance of mutualistic plant–microbial interactions in the rhizosphere and within plant roots involve several root cell types. Major cell types are the peripheral epidermis, often including both root hair-developing trichoblasts as well as atrichoblasts, the cortex including the endodermis as its innermost cell layer, the pericycle, and the central vasculature with phloem and xylem elements for rootward and shootward long-distance transport, respectively [6] (Fig. 1). These distinct cell types differentially contribute to the functionality of the organ and plant as a whole [1, 4]. Beyond differential patterns of transcriptional and/or translational activity, the coordination of developmental processes requires intercellular communication and signal transduction between root cell types, in which diverse molecules including hormones [11,12,13,14], peptides [15,16,17] and regulatory RNAs [17, 18] are involved

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