SrSymRK, a plant receptor essential for symbiosome formation

Abstract

The symbiosis between legumes and rhizobia is essential for the nitrogen input into the life cycle on our planet. New root organs, the nodules, are established, which house N2-fixing bacteria internalized into the host cell cytoplasm as horizontally acquired organelles, the symbiosomes. The interaction is initiated by bacterial invasion via epidermal root hair curling and cell division in the cortex, both triggered by bacterial nodulation factors. Of the several genes involved in nodule initiation that have been identified, one encodes the leucine-rich repeat-type receptor kinase SymRK. In SymRK mutants of Lotus japonicus or its orthologs in Medicago sp. and Pisum sativum, nodule initiation is arrested at the level of the root hair interaction. Because of the epidermal block, the role of SymRK at later stages of nodule development remained enigmatic. To analyze the role of SymRK downstream of the epidermis, the water-tolerant legume Sesbania rostrata was used that has developed a nodulation strategy to circumvent root hair responses for bacterial invasion. Evidence is provided that SymRK plays an essential role during endosymbiotic uptake in plant cells.

Fig. 1.

SrSymRK, the SymRK ortholog of S. rostrata.(A) Sequence of the SrSymRK protein and orthologs. SrSymRK shows a 90% and 86% similarity to L. japonicus LjSymRK and M. truncatula MtDMI2, respectively. Conserved amino acids are highlighted. (B) Southern analysis with the extracellular domain of SrSymRK as a probe.

Fig. 2.

Expresssion analysis of SrSymRK. (A) RT-PCR analysis. SrSymRK expression levels are shown for leaves, flowers, uninfected adventitious root primordia (primordium), in adventitious root primordia infected with ORS571 after 2, 4, and 6 dpi, in hydroponic roots (T^-), and in hydroponic roots 12, 24, 36, 72, and 84 h after inoculation. (B) Bright-field image of an in situ mRNA localization in a semithin section through a nodule primordium 72 h after inoculation. (C) Dark-field image of B; signal is visible as silver grains in the infection zone. ip, infection pocket; iz, infection zone; m, meristem. (Scale bars, 100 μm.)

Fig. 3.

Knockdown phenotype of SrSymRK.(A) Low magnification view of a SrSymRK knockdown line, 10 dpi. Bacteria are labeled with β-glucuronidase. (B) Low magnification view of a functional WT nodule, 10 dpi. Bacteria are labeled with β-glucuronidase. (C) Semithin section through A. IPs, ITs, and a primordium are formed, but no cells are infected. (D) Semithin section through B, 10 dpi. (E and F) Details of C and D. Uninfected and infected cells are marked by triangles and asterisks, respectively. ct, central tissue; ip, infection pocket; it, infection thread; iz, infection zone; m, meristem. (Scale bars, 100 μm.)

Fig. 4.

Ultrastructural analysis of the SrSymRK knockdown phenotype. (A) Transverse section through a WT intracellular IT. (B) Transverse section through an intracellular IT in an SrSymRK knockdown background, 10 dpi. Arrowhead and arrow indicate low electron-dense rim and abnormal cell wall depositions in SrSymRK knockdown lines, respectively. (C) Section through a bag-like intracellular IT consisting of numerous branches, 10 dpi. (D) Section through an intracellular IT in an SrSymRK knockdown background. Low electron-dense rim is indicated by an arrowhead. (E) Rare release of bacteria, resulting in abnormal symbiosomes, which are indicated by arrows. (F) WT bacteroids at 6 dpi. (Scale bars, 1 μm.)