Regulatory roles of small RNAs in nodulation symbiosis
Katharina Markmann, PhD
Junior Group Leader
Zentrum für Molekularbiologie der Pflanzen
Auf der Morgenstelle 32
72076 Tübingen, Germany
Tel.: 0049 (0) 7071 29 73222
Dissecting small RNA involvement in symbiosis signalling and regulation
Legumes have evolved the ability to respond to nitrogen–fixing bacteria termed rhizobia by forming root nodules. Within these symbiotic organs, the bacteria reside intra-cellularly, making reduced atmospheric nitrogen available to the plant in exchange for carbohydrates and other nutrients.
The small, transformable host Lotus japonicus (Lotus) and its rhizobial symbiont Mesorhizobium loti represent an attractive model system for root endosymbiosis, and an extensive pool of mutant, sequence and software resources became available in recent years.
Several plant genes have been identified that are essential in mediating responses to rhizobial signaling molecules including chitooligosaccharide nodulation (Nod) factors and exopolysaccharides. Despite this, the current understanding of plant responses to symbiotic stimulation remains skeletal. In particular, regulatory links between known signaling components are often unclear.
In both plants and animals, small RNA (sRNA) guided control of transcript and protein abundance has emerged as a key regulatory mechanism. Apart from affecting core developmental and physiological processes, sRNA directed silencing plays an essential role in plant responses to biotic and abiotic stresses.
Several miRNAs have been functionally associated with nodulation symbiosis, or shown to regulate symbiosis-related transcription factors (for a review, see Lelandais-Brière et al., 2016). Our research aims to gain insights into the roles of small RNAs (sRNAs) in root endosymbioses, and promote the understanding of these interactions by isolating and functionally characterizing relevant sRNA-target nodes, with a particular interest in miRNAs involved in integrating symbiosis and plant development. A recent focus of our investigations lies on roles of miRNAs in systemic auto-control of symbiosis involving shoot-root crosstalk.
Identification and analysis of sRNA-target nodes in symbiosis
On the basis of an extensive sRNA sequencing effort featuring Lotus mutants impaired at different stages of nodulation symbiosis, we selected candidate sRNAs with potential involvement in symbiosis related signalling. To streamline the analysis of this as well as follow-up datasets, we established a bioinformatic pipeline, shortran (Gupta et al., 2012), for comparative profiling and annotation of sRNA deep sequencing data. This modular analysis tool with an easily searchable output in MySQL (www.mysql.com) database format will also form the basis for analyzing sRNA seq libraries sequenced as part of the project proposed here.
With a particular focus on miRNAs, our ongoing efforts focus on sRNA candidates emerging from these datasets and their involvement in three aspects of nodulation symbiosis:
(a) epidermal infection,
(b) nodule development, infection and function and
(c) systemic control of symbiosis.
Making use of the extensive Lotus mutant resources available, we further analyze the role of small RNA role of genes involved in sRNA production and function in Lotus symbiosis (d).
(a) sRNA regulation of epidermal infection
As sRNAs are fast-evolving molecules that can significantly alter plant response patterns to external stimuli, we saw potential for their involvement also in the earliest symbiotic responses. In a study featured in Holt et al. (2015), we used Illumina deep sequencing to trace sRNA dynamics during the first steps of legume interactions with rhizobial bacteria in wild type plants. This dataset revealed 226 novel Lotus miRNAs and 76 infection-responsive sRNAs and presents a rich resource for ongoing and future investigations. A follow-up dataset making use of five mutant lines impaired at distinct stages of epidermal infection is under analysis. Insights from these datasets suggest that regulatory sRNAs, and miRNAs in particular, play an important role in controlling and fine-tuning gene regulation during legume infection with rhizobial bacteria (Holt et al., 2015). Several infection responsive miRNAs, such as miR172, belong to families implicated in conserved developmental processes, indicating that symbiosis evolution involved functional diversification of miRNA directed gene regulation. Interestingly, our data suggests that the majority of alterations in small RNAs were attributed to symbiosis-responsive miRNAs.
In an effort to provide a toolset for higher-resolution functional analysis of the candidate sRNA-target nodes retrieved from the datasets outlined in (a) and (b), we identified a series of promoters with activity limited to or highly enriched in one or few Lotus root or nodule cell types (Gavrilovic et al., 2016). Major root cell types covered by these promoters include epidermis, inner cortex, pericycle and endodermis, phloem or xylem. These promoters are an important asset to functional analyses of candidate miRNAs and localization of miRNA-target node activity in the context of the project proposed here.
(b) sRNAs regulating nodule development and function
To dissect sRNA involvement in later stages of nodulation symbiosis, we sequenced an additional set of sRNA libraries covering nodules of Lotus wild type and three mutant lines. The latter include two lines carrying mutations in independent genes involved in micronutrient transport to bacteroids, the symbiotic stage of rhizobial bacteria within nodules. Both stationary endosymbiont nodule (sen)1 and symbiotic sulphate transporter (sst)1 loss-of-function mutants form infected but non-functional nodules that are unable to make reduced nitrogen available to the host. In addition, we examined spontaneous, bacteria-free nodule organs formed on spontaneous nodule formation (snf)1 mutants. These datasets will serve as a valuable reference providing insights in potential involvement of candidate sRNAs in different stages of symbiosis development.
To aid miRNA target verification, we further generated a degradome (PARE) sequencing dataset revealing mRNAs that undergo sRNA-mediated endonucleolytic cleavage during the stages of epidermal invasion and nodulation. These data will be instrumental for the functional analysis of sRNA candidates emerging from the sequencing approach proposed for this project. Manuscripts presenting these combined datasets are in preparation.
(c) sRNAs in the systemic control of symbiosis
Several potentially infection-related candidate sequences emerging from our sRNA sequencing datasets belong to conserved miRNA families that are thought to involve in systemic nutrient homeostasis in other species, including Arabidopsis. To ensure a viable nutrient balance in the face of bacterial interactors that fully rely on plant provided resources, host plants must systemically integrate nutrient homeostasis and symbiosis progression to maintain a fitness-enhancing mutualistic association and prevent it from tilting towards pathogenicity. Consistently, nodulation symbiosis relies on a systemic autocontrol mechanism termed autoregulation of nodulation (AON). Mutant hosts impaired in AON show hypersymbiosis phenotypes in the form of an overabundance of infection and/or nodulation events.
As sRNAs are capable of cell-to-cell as well as systemic movement in plants, and act at a high level of specificity, we consider them interesting candidate factors in systemic symbiosis control.
(d) The Lotus japonicus small RNA machinery
To understand the role of regulatory small RNAs in root symbiosis, we have identified loss-of-function mutants in a number of genes that are potentially necessary for small RNA production and activity in the cell. We are examining these mutants and evaluate their ability to interact with nitrogen fixing rhizobial bacteria as well as mycorrhizal fungi. Defects or differences in symbiosis development in these mutants compared to genetically healthy wild type plants where small RNA regulation is intact help us identify the roles the respective genes, and their associated small RNAs, play in these processes.