Nitrogen source interacts with ROP signalling in root hair tip‐growth

Root hairs elongate in a highly polarized manner known as tip growth. Overexpression of constitutively active Rho of Plant (ROP)/RAC GTPases mutants induces swelling of root hairs. Here, we demonstrate that Atrop11^CA‐induced swelling of root hairs depends on the composition of the growth medium. Depletion of ammonium allowed normal root hair elongation in Atrop11^CA plants, induced the development of longer root hairs in wild‐type plants and suppressed the effect of Atrop11^CA expression on actin organization and reactive oxygen species distribution, whereas membrane localization of the protein was not affected. Ammonium at concentrations higher than 1 mM and the presence of nitrate were required for induction of swelling. Oscillations in wall and cytoplasmic pH are known to accompany tip growth in root hairs, and buffering of the growth medium decreased Atrop11^CA‐induced swelling. Fluorescence ratio imaging experiments revealed that in wild‐type root hairs, the addition of NH₄NO₃ to the growth medium induced an increase in the amplitude of extracellular and intracellular pH oscillations and an overall decrease in cytoplasmic pH at the cell apex. Based on these results, we suggest a model in which ROP GTPases and nitrogen‐dependent pH oscillations function in parallel pathways, creating a positive feedback loop during root hair growth.     

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–Rhizobium Symbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.     

Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.     

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.     

The small GTPase ROP6 interacts with NFR5 and is involved in nodule formation in Lotus japonicus

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.     

Rearrangement of Actin Cytoskeleton Mediates Invasion of Lotus japonicus Roots by Mesorhizobium loti

Infection thread–dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

Calcium Spiking Patterns and the Role of the Calcium/Calmodulin-Dependent Kinase CCaMK in Lateral Root Base Nodulation of Sesbania rostrata

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca²⁺ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca²⁺ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca²⁺ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca²⁺ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca²⁺ spiking decoding.     

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.     

A mutation in MRH2 kinesin enhances the root hair tip growth defect caused by constitutively activated ROP2 small GTPase in Arabidopsis

Root hair tip growth provides a unique model system for the study of plant cell polarity. Transgenic plants expressing constitutively active (CA) forms of ROP (Rho-of-plants) GTPases have been shown to cause the disruption of root hair polarity likely as a result of the alteration of actin filaments (AF) and microtubules (MT) organization. Towards understanding the mechanism by which ROP controls the cytoskeletal organization during root hair tip growth, we have screened for CA-rop2 suppressors or enhancers using CA1-1, a transgenic line that expresses CA-rop2 and shows only mild disruption of tip growth. Here, we report the characterization of a CA-rop2 enhancer (cae1-1 CA1-1) that exhibits bulbous root hairs. The cae1-1 mutation on its own caused a waving and branching root hair phenotype. CAE1 encodes the root hair growth-related, ARM domain-containing kinesin-like protein MRH2 (and thus cae1-1 was renamed to mrh2-3). Cortical MT displayed fragmentation and random orientation in mrh2 root hairs. Consistently, the MT-stabilizing drug taxol could partially rescue the wavy root hair phenotype of mrh2-3, and the MT-depolymerizing drug Oryzalin slightly enhanced the root hair tip growth defect in CA1-1. Interestingly, the addition of the actin-depolymerizing drug Latrunculin B further enhanced the Oryzalin effect. This indicates that the cross-talk of MT and AF organization is important for the mrh2-3 CA1-1 phenotype. Although we did not observe an apparent effect of the MRH2 mutation in AF organization, we found that mrh2-3 root hair growth was more sensitive to Latrunculin B. Moreover, an ARM domain-containing MRH2 fragment could bind to the polymerized actin in vitro. Therefore, our genetic analyses, together with cell biological and pharmacological evidence, suggest that the plant-specific kinesin-related protein MRH2 is an important component that controls MT organization and is likely involved in the ROP2 GTPase-controlled coordination of AF and MT during polarized growth of root hairs.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.     

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.