Lotus japonicus natural diversity unveils genetic components to host rhizobia inside plant cells

Espejel Venado, Oscar Rafael (2022): Lotus japonicus natural diversity unveils genetic components to host rhizobia inside plant cells. Dissertation, LMU München: Fakultät für Biologie

Abstract

Legume plants form a symbiosis with nitrogen-fixing rhizobia bacteria. This symbiosis occurs within cells of specialized root organs called nodules in which a bidirectional nutrient exchange between the symbionts takes place. During this process, legumes obtain reduced nitrogenated compounds whereas rhizobia receive carbon compounds derived from plant photosynthesis. Therefore, there has been great interest in unveiling the genetic architecture of this phenomenon to reduce the use of inorganic nitrogen fertilizers in agriculture. The study of root nodule symbiosis has determined the function of nearly 200 genes in the last 20 years. This has been possible through forward and reverse genetic screenings and the development of various other genomic tools in model organisms such as Lotus japonicus (L. japonicus). Gene discovery using conventional screenings has almost reached its limit. Thus, new approaches are needed to uncover new genetic players, particularly genes involved in the plant cell mechanisms required to host bacteria inside the plant cells. Molecular understanding of the tissue and cellular adaptations required to host rhizobia inside nodules remains extremely limited, because of the difficulty in disconnecting nodule formation from infection. These modifications provide an adequate environment for effective nitrogen fixation. A key modification is the development of a cell layer that surrounds the inner cells and reduces the amount of oxygen that enters these cells, thus, protecting the bacterial nitrogenase, which is oxygen-labile. However, the genetic components to form this barrier are still unknown. Recently, Liang et al. characterized a system that opens up the possibility of studying the mechanism of internalization of the bacteria inside the plant cells and by consequence the bacterial accommodation. This system describes the interaction between a Lotus species and a subcompatible rhizobium strain, Rhizobium leguminosarum (R. leguminosarum) Norway. In this system, the bacteria enter the plant cell without the aid of specialized structures in the root hair called infection threads but rather from an alternative mechanism of infection independent of these threads. In this work, this system was expanded by exploring the natural diversity of different L. japonicus accessions in combination with R. leguminosarum Norway. By using this approach, the nodule organogenesis and infection programs were uncoupled, as it induces nodules that remain uninfected in some L. japonicus accessions. Comparative transcriptomic analysis of infected and uninfected nodules yielded 167 differentially regulated genes. Among these, genes with functions associated with plant barrier formation were specifically upregulated in infected nodules. Among the genes uncovered, two fatty acyl-CoA reductases (FARs) genes that are involved in the production of cuticular waxes, seed coat, bundle sheath, bark tissues and two putative scaffold proteins Casparian Strip Domain-Like Proteins (CASPLs) were studied. It was hypothesized that these genes are involved in the formation of a cellular barrier that controls the delicate oxygen homeostasis in the root nodule. Spatiotemporal analysis of promoter activity controlling the expression of FARs and CASPLs revealed tissue-specific activation in the nodule endodermis and infected cells, respectively. Reverse genetic analysis was performed by investigating two Lotus retrotransposon lines (LORE1) in a nodule specific FAR and a double mutant in a CASPL generated by CRISPR-Cas12a editing. In the first case, mutants compared to wild-type nodules displayed a significant reduction in hydrophobic polyesters in the surrounding cell layer termed nodule endodermis, an increase in their oxygen concentration inside the mutant nodules, and impaired nitrogen fixation activity. This transduced in mutant plants having significantly shorter shoots. These results support a model in which disruption in the composition of the nodule oxygen barrier alters nitrogen fixation. In the second case, infected nodule cells in the double mutant line showed an irregular morphology with an undefined nucleus compared with wild-type cells. This suggests that local cell wall modifications are required to properly accommodate the symbiont. These results pave the way for understanding how plants modify their cell walls locally to host the symbiont.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. August 2022
• 1. Bericht­erstatter:in: Marín, Macarena