Calcium induces the cleavage of NopA and regulates the expression of nodulation genes and secretion of T3SS effectors in Sinorhizobium fredii NGR234

Authors: KIM, WONSEOK - University Of Missouri; ACOSTA-JURADO, SEBASTIAN - Pablo De Olavide University; KIM, SUNHYUNG - University Of Missouri; Krishnan, Hari

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2024
Publication Date: 3/19/2024
Citation: Kim, W., Acosta-Jurado, S., Kim, S., Krishnan, H.B. 2024. Calcium induces the cleavage of NopA and regulates the expression of nodulation genes and secretion of T3SS effectors in Sinorhizobium fredii NGR234. International Journal of Molecular Sciences. 25(6). Article 3443. https://doi.org/10.3390/ijms25063443.
DOI: https://doi.org/10.3390/ijms25063443

Interpretive Summary: Legumes such as soybeans have a unique ability to combine beneficially with specialized soil bacteria collectively known as rhizobia. Nodules are a specialized plant organ in which atmospheric nitrogen is reduced to ammonia by the enzyme nitrogenase encoded by rhizobia. This process is called biological nitrogen fixation and contributes significantly to the overall nitrogen needs of plants. Several environmental factors, including soil calcium content, can regulate biological nitrogen fixation. Despite the important role of calcium in nodulation, little is known about the molecular mechanisms by which calcium controls nodulation. In this study, we investigated the effect of calcium on global rhizobium gene expression by performing RNA sequencing experiments. Our results showed that calcium altered the expression of several important genes controlling nodulation. The information obtained from this study will help scientists better understand the factors that limit the formation of nitrogen-fixing nodules on legumes. Such an understanding would allow scientists to manipulate biological nitrogen fixation so that American farmers could increase yields with minimal use of nitrogen fertilizers.

Technical Abstract: The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia–legume interactions.