Laser Microdissection of Pisum sativum L. Nodules Followed by RNA-Seq Analysis Revealed Crucial Transcriptomic Changes during Infected Cell Differentiation

Abstract

Garden pea (Pisum sativum L.) is a globally important legume crop. Like other legumes, it forms beneficial symbiotic interactions with the soil bacteria rhizobia, gaining the ability to fix atmospheric nitrogen. In pea nodules, the meristem is long-lasting and results in the formation of several histological zones that implicate a notable differentiation of infected host cells. However, the fine transcriptional changes that accompany differentiation are still unknown. In this study, using laser microdissection followed by RNA-seq analysis, we performed transcriptomic profiling in the early infection zone, late infection zone, and nitrogen fixation zone of 11-day-old nodules of pea wild-type line SGE. As a result, a list of functional groups of differentially expressed genes (DEGs) in different nodule histological zones and a list of genes with the most prominent expression changes during nodule development were obtained. Their analyses demonstrated that the highest amount of DEGs was associated with the nitrogen fixation zone. Among well-known genes controlling nodule development, we revealed genes that can be novel players throughout nodule formation. The characterized genes in pea were compared with those previously described in other legumes and their possible functions in nodule development are discussed.

A variable gene family encoding nodule-specific cysteine-rich peptides in pea (Pisum sativum L.)

“…For all identified NCR genes, the analysis of spatiotemporal expression profiles was carried out using data of 3′ MACE sequencing of P. sativum wild-type nodules (SGE line) at 12, 21, 28, and 42 dpi and data of RNAseq obtained from microdissected nodules (early zone II, late zone II, and zone III) of the same SGE line at 11 dpi (for a description of methods, see Kusakin et al, 2021 ).…”

“…For analysis of the spatial expression patterns of NCR genes, RNA sequencing data from the nodule microdissection experiment were used ( Kusakin et al, 2021 ). Based on NCR gene expression levels in the early infection zone (zone early II), late infection zone (zone late II), and nitrogen fixation zone (zone III) of the nodule, two main clusters were revealed—a maximum of expression in late II zone and a maximum in zone III ( Figure 5B ).…”

Improving Bambara Groundnut Production: Insight Into the Role of Omics and Beneficial Bacteria

“…The influence of plant growth-promoting microbes on the nodule and its bacteroid transcriptomes have been reported ( Kelly et al, 2018 ; Lamouche et al, 2019 ). Researchers have employed laser-capture microdissection coupled with RNA sequencing for the total elucidation of expression patterns throughout the nodule compartment ( Kusakin et al, 2021 ; Li et al, 2021 ). This technology is promising in fully understanding the gene expression patterns in nodules in response to microbe colonization.…”