RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections
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eLife assessment
This work, which will be of interest to all who study plant-microbe interactions as well as plant cell biology, addresses a fundamental question in symbiosis, placing a classic nodulation defective mutant (rpg) into a plausible protein complex and establishing a hierarchy of "infectosome" assembly. Evidence includes convincing genetics and subcellular localization of components during establishment and maintenance of infection. The study also includes compelling new FLIM-based imaging techniques to distinguish signals from closely associated domains in plant cells.
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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 the 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.
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eLife assessment
This work, which will be of interest to all who study plant-microbe interactions as well as plant cell biology, addresses a fundamental question in symbiosis, placing a classic nodulation defective mutant (rpg) into a plausible protein complex and establishing a hierarchy of "infectosome" assembly. Evidence includes convincing genetics and subcellular localization of components during establishment and maintenance of infection. The study also includes compelling new FLIM-based imaging techniques to distinguish signals from closely associated domains in plant cells.
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Reviewer #1 (Public Review):
RPG was identified as a mutant with poor nodulation in 2008, but its activity was unclear. Interest in its role was rejuvenated by a phylogenetic approach where RPG was one of only three genes that have been consistently lost in non-nodulating species in the Fagales/Fabales/Cucurbitales/Rosales families. In this careful genetic and imaging-based analysis of the role of the RPG gene in nodulation, the authors use transient and stable genetic transformation in nodulating Medicago roots to clarify the subcellular localization of RPG and its relationship to infection thread maintenance along with two other better characterized symbiosis proteins VAPYRIN (VPY) and LUMPY INFECTION (LIN). Detailed phenotypic analysis of two RPG mutants indicates that RPG is required to sustain polarization of the membrane at the …
Reviewer #1 (Public Review):
RPG was identified as a mutant with poor nodulation in 2008, but its activity was unclear. Interest in its role was rejuvenated by a phylogenetic approach where RPG was one of only three genes that have been consistently lost in non-nodulating species in the Fagales/Fabales/Cucurbitales/Rosales families. In this careful genetic and imaging-based analysis of the role of the RPG gene in nodulation, the authors use transient and stable genetic transformation in nodulating Medicago roots to clarify the subcellular localization of RPG and its relationship to infection thread maintenance along with two other better characterized symbiosis proteins VAPYRIN (VPY) and LUMPY INFECTION (LIN). Detailed phenotypic analysis of two RPG mutants indicates that RPG is required to sustain polarization of the membrane at the advancing tip of the IT, and without RPG, the plant microtubule cytoskeleton organization, nuclear position, and localization of VPY and the cell wall modifying enzyme NODULE PECTATE LYASE all differ from normal.
The model that emerges and that is well supported by the presented data is that RPG behaves as a scaffold to convert/customize the cellular machinery that organizes cytoskeleton and membrane into something that facilitates infection thread polarity. This work established a hierarchy of "infectosome" assembly using genetics and localization of components.
The strengths are the use of new imaging modalities to define the location and functional dependencies of complex components. This paper makes numerous significant advances in the understanding of infection thread maintenance and assigning a cellular activity for RPG. It leaves open still questions about how RPG is localized to discrete puncta and how it recruits other proteins to these still symbiosis-related structures.
This is an exciting addition to the literature detailing the mechanisms by which host plant cells make environments hospitable to symbionts, the strong correlation with the presence/absence of RPG and the ability/failure to support nodulation in a variety of plant species makes RPG an attractive candidate for engineering nodulation in crop plants that currently lack it.
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Reviewer #2 (Public Review):
The infection thread is a polarized structure with tip growth, much like the root hair itself, but which grows inward. To understand this unique and important structure, forward genetics approaches have been carried out, and have uncovered a number of seemingly disparate factors. Recently evidence has emerged that these proteins physically collaborate to tie together vesicle trafficking, cytoskeleton, and the nucleus. This study not only shows that RPG is part of this protein complex, but likely an organizer of this complex through its CC domains. The authors methodically dissected the behavior of this protein, and used cutting-edge microscopical tools to show that this protein is required for infectosome formation and the recruitment of a specific phospholipid to the tip of the IT, which are in turn …
Reviewer #2 (Public Review):
The infection thread is a polarized structure with tip growth, much like the root hair itself, but which grows inward. To understand this unique and important structure, forward genetics approaches have been carried out, and have uncovered a number of seemingly disparate factors. Recently evidence has emerged that these proteins physically collaborate to tie together vesicle trafficking, cytoskeleton, and the nucleus. This study not only shows that RPG is part of this protein complex, but likely an organizer of this complex through its CC domains. The authors methodically dissected the behavior of this protein, and used cutting-edge microscopical tools to show that this protein is required for infectosome formation and the recruitment of a specific phospholipid to the tip of the IT, which are in turn necessary for polar growth. The data are largely convincing, and the claims are justified. However, a number of their main findings need to be better explained.
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Reviewer #3 (Public Review):
The article reports the functional analysis of one of the critical genetic determinants for bacterial infection, the RHIZOBIUM-DIRECTED POLAR GROWTH protein (RPG). The evolutionary pattern linking RPG to the Transcription Factor and the LysM receptor-like kinase, and the ability to form root nodule symbiosis makes it one of the prime candidates for engineering symbiotic nitrogen fixation in cereals. Therefore this analysis is timely and of huge importance. In a previous study, an EMS-induced mutant of M. truncatula (rpg-1 allele) was reported to have aberrant infection threads and poorly colonized nodules, the RPG expression was strongly associated with rhizobial infection, and the RPG protein showed nuclear localization when heterologously overexpressed in N. benthamiana (Arrighi et al., 2008), but its …
Reviewer #3 (Public Review):
The article reports the functional analysis of one of the critical genetic determinants for bacterial infection, the RHIZOBIUM-DIRECTED POLAR GROWTH protein (RPG). The evolutionary pattern linking RPG to the Transcription Factor and the LysM receptor-like kinase, and the ability to form root nodule symbiosis makes it one of the prime candidates for engineering symbiotic nitrogen fixation in cereals. Therefore this analysis is timely and of huge importance. In a previous study, an EMS-induced mutant of M. truncatula (rpg-1 allele) was reported to have aberrant infection threads and poorly colonized nodules, the RPG expression was strongly associated with rhizobial infection, and the RPG protein showed nuclear localization when heterologously overexpressed in N. benthamiana (Arrighi et al., 2008), but its cellular and molecular function in M. truncatula have not been understood in detail.
In the present manuscript, the authors showed that RPG is a crucial component of the infectosome machinery. They conclude that RPG sustains polar growth of intracellular infection threads, being necessary to recruit via protein-protein interactions the VAPYRIN.
In absence of rpg, the authors reported : (i) the absence of membrane polarization at the advancing tip of the infection thread using phosphoinositide reporters, (ii) the lack of connectivity between the infection thread tip and the nucleus via the microtubules cytoskeleton, (iii) the loss of polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL).These results confirmed that RPG is part of the core host machinery required to support symbiont accommodation. Furthermore, this work shows that multimeric host factors work as a module committed to endosymbiosis to sustain the infection thread-mediated bacterial infection.
This paper is well written, with extremely beautiful cell biology and the conclusions are clearly connected to the data presented. However, in my view, some critical points need to be further addressed:
1. The author showed that RPG co-purified with EXO70H4 and VPY indicating that these proteins indeed belong to the same complex. Moreover, they showed that the major molecular determinant conferring functionality to RPG resides in its coiled-coil domain with structure-function analysis. Does the CC domain important for RPG interaction with "the multimeric host factors" described here? What is the localization of the truncated form of RPG (reported in this paper for rpg1 complementation analysis) in the WT plant?
2. It is not clear to me how RPG acts on the membrane polarity: Is there a direct role of RPG in the PIP2 polarization, and cytoskeleton arrangement in the exocytosis of cell wall modifying enzyme NODULE PECTATE LYASE (NPL)? Did the authors observe an increase in membrane polarity when RPG is overexpressed? The authors might consider adding a model for the different molecular components they studied in the context of this biotic interaction.
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