RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections

Abstract

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular structures (infection threads - ITs) that extend across cell layers via polar tip-growth. Comparative phylogenomic studies have identified RPG (RHIZOBIUM-DIRECTED POLAR GROWTH) among the critical genetic determinants for bacterial infection. In Medicago truncatula, RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remains elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co-localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment into these structures. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection.

Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes

“… 37 ) and CYCLOPS / IPD3 (ref. 38 ), the master symbiotic regulator NIN 39 or the infection-associated gene RPG 40 , 41 . The phylogenetic distribution of three of them ( LjNFR5/MtNFP , NIN and RPG ) has been recently linked with the ability to form RNS in the NFN clade.…”

Competence for transcellular infection in the root cortex involves a post-replicative, cell-cycle exit decision inMedicago truncatula

“…In summary, we hypothesize that endocycle suppression after a round of DNA replication (Breakspear et al, 2014) and exiting the cell-cycle from the G2-phase might therefore create a permissive, yet controlled state for cortical infection thread progression where polarization and cell-wall remodelling factors specialized for intracellular infection (Lace et al, 2023; Su et al, 2023) act in a defined cell-cycle window to sustain infection thread growth and guidance across cortical layers.…”

“…The conversion of atmospheric dinitrogen into a usable form is highly energy-demanding and requires a low-oxygen environment, which is provided within nodules, specialized lateral organs formed on the roots that are densely populated by rhizobia. A widely adopted mechanism to access the developing nodule cells is the formation of cortical infection threads, transcellular conduits formed upon the invagination of the host plasma membrane and hijacking the cellular machinery of infected root cortical cells (Timmers et al, 1999;Su et al, 2023).…”

Annexin- and calcium-regulated priming of legume root cells for endosymbiotic infection

“…ITs are formed in root hairs after rhizobia have been entrapped and enclosed in a globular infection chamber, a process orchestrated by dynamic changes in the cytoskeleton-plasma-membrane-cell wall interface 24–26 . ITs grow polarly from cell to cell towards the developing nodule forming underneath 27,28 , and these two programs must be tightly synchronized before a fully functional nodule is formed under the influence of key plant hormones, intercellular signalling and long-distance regulatory pathways 29–32 .…”