Legume-rhizobia symbiosis: Translatome analysis

Abstract

Leguminous plants can establish endosymbiotic relationships with nitrogen-fixing soil rhizobacteria. Bacterial infection and nodule organogenesis are two independent but highly coordinated genetic programs that are active during this interaction. These genetic programs can be regulated along all the stages of gene expression. Most of the studies, for both eukaryotes and prokaryotes, focused on the transcriptional regulation level determining the abundance of mRNAs. However, it has been demonstrated that mRNA levels only sometimes correlate with the abundance or activity of the coded proteins. For this reason, in the past two decades, interest in the role of translational control of gene expression has increased, since the subset of mRNA being actively translated outperforms the information gained only by the transcriptome. In the case of legume-rhizobia interactions, the study of the translatome still needs to be explored further. Therefore, this review aims to discuss the methodologies for analyzing polysome-associated mRNAs at the genome-scale and their contribution to studying translational control to understand the complexity of this symbiotic interaction. Moreover, the Dual RNA-seq approach is discussed for its relevance in the context of a symbiotic nodule, where intricate multi-species gene expression networks occur.

Figure 1 -. Main events occurring during the initial stages of legume-rhizobia symbiosis. The interaction initiates at the root hairs, where the exchange of molecular signals - plant flavonoids and rhizobia Nod factors (NF) - occurs. When the interaction between the symbiotic pair is effective, the rhizobia adheres to the root hair, which curls, entrapping the bacterium and forming an infectious focus. Then, an infection thread is formed due to cell wall degradation and invagination of the root hair plasma membrane. This tubular structure progresses, allowing the rhizobia to infect the root cortex, where the activation of cortical cell division originates the nodule meristem (nodule organogenesis). The two highly coordinated genetic programs activated during legume-rhizobia symbiosis are bacterial infection and cortex cell division. The first one takes place at the epidermis and involves the perception of NF through NF receptors, calcium oscillations, perception of the calcium oscillations via calcium-activated kinase (CCaMK), and transcription factors (NSP, NIN, ERN1) leading to gene expression activation. An increase in cytokinin levels promotes cortical cell division, where the signaling pathway that involves the cytokinin receptor LHK1/CRE1 and transcription factors such as NSP1/2 and NIN, among others, leads to nodule organogenesis.

Figure 2 -. Schematic overview of the main tools for mRNA translation analysis at the genome-scale in the plant field. After plant tissue collection and flash freezing, translation is halted with cycloheximide (CHX), and a cell lysate is obtained. The cytoplasmic lysate containing polysomes, monosomes, and ribosomal subunits could be used for further ribosome-associated RNA isolation following different techniques: 1) Polysome Profiling is a technique that separates actively translating mRNAs using sucrose density gradient ultracentrifugation. Once the polysome fraction is obtained, Trizol RNA isolation and conversion into cDNA libraries are performed for RNA sequencing (RNA-seq). 2) TRAP (Translating Ribosome Affinity Purification) is based on the epitope tagging of a ribosomal protein for the immunopurification of ribosome-mRNA complexes. Cytoplasmic lysate from transgenic plants with ribosomal protein tagging undergoes immunoprecipitation with anti-tag antibodies to isolate the polysomal fraction, followed by Trizol RNA isolation and conversion into cDNA libraries for performing RNA-seq. 3) Ribosome Profiling is a technique that treats cytoplasmic lysates with RNA-digesting enzymes to degrade the ribosome-unprotected mRNAs. After RNase digestion, monosomes are isolated, and ribosome-protected mRNA fragments (footprints) are purified and converted into cDNA libraries for RNA-seq.

Figure 3 -. Schematic representation of the Dual-Seq protocol for plant-rhizobium symbiotic samples, including wet lab and the basic data analysis workflow. Fresh symbiotic nodules are detached from the plant roots, flash-frozen, and stored at -80 °C until used. Long storage periods are detrimental both for eukaryotic and prokaryotic RNAs but mainly for the prokaryotic ones. After total RNA extraction, quantification and integrity evaluation of the samples is performed. In the ribosomal RNA (rRNA) depletion step, each sample is split in two to perform prokaryotic RNA enrichment along with the rRNA depletion. Once the two previously split samples are pooled, cDNA sequencing libraries are made, followed by high throughput NGS sequencing. The basic data analysis workflow comprises visual quality inspection, followed by adapter and low-quality bases trimming. Then, reads are mapped to an unified (i.e. plant + symbiont) reference genome, followed by per gene read quantification and normalization. Differential gene expression (DEG) analysis, pathway enrichment analysis, co-expression network analysis, and comparative transcriptomic, among others, can be performed to interpret the data.