Antonius C.J. Timmers

A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca2+ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca2+ spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca2+ responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca2+ spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca2+ oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca2+ spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca2+ concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca2+ oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Mechanism of Infection Thread Elongation in Root Hairs of Medicago truncatula and Dynamic Interplay with Associated Rhizobial Colonization

In temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). To study IT development in living root hairs, a protocol has been established for Medicago truncatula that allows confocal microscopic observations of the intracellular dynamics associated with IT growth. Fluorescent labeling of both the IT envelope (AtPIP2;1-green fluorescent protein) and the host endoplasmic reticulum (green fluorescent protein-HDEL) has revealed that IT growth is a fundamentally discontinuous process and that the variable rate of root hair invagination is reflected in changes in the host cell cytoarchitecture. The concomitant use of fluorescently labeled Sinorhizobium meliloti has further revealed that a bacteria-free zone is frequently present at the growing tip of the IT, thus indicating that bacterial contact is not essential for thread progression. Finally, these in vivo studies have shown that gaps within the bacterial file are a common feature during the early stages of IT development, and that segments of the file are able to slide collectively down the thread. Taken together, these observations lead us to propose that (1) IT growth involves a host-driven cellular mechanism analogous to that described for intracellular infection by arbuscular mycorrhizal fungi; (2) the non-regular growth of the thread is a consequence of the rate-limiting colonization by the infecting rhizobia; and (3) bacterial colonization involves a combination of bacterial cell division and sliding movement within the extracellular matrix of the apoplastic compartment.

Two novel proteins, PopB, which has functional nuclear localization signals, and PopC, which has a large leucine‐rich repeat domain, are secreted through the Hrp‐secretion apparatus of Ralstonia solanacearum

The Ralstonia solanacearum hrp gene cluster codes for components of a type III secretion pathway necessary for the secretion of PopA1, a hypersensitive response‐like elicitor protein. In the present study, we show that several other Hrp‐secreted proteins can be detected by growing wild‐type bacteria in minimal medium in the presence of Congo red. Two of these proteins, PopB and PopC, are encoded by genes located downstream of popA and constitute an operon with popA. popABC mutants retain the wild‐type ability to cause disease in hosts and to elicit the hypersensitive response on non‐hosts. Expression of the popABC operon is controlled by the hrpB regulatory gene and is induced upon co‐culture with Arabidopsis cell suspensions. This plant cell‐specific induction depends on PrhA, a putative receptor for plant specific signal(s). The transcription of the popABC operon is not modified by the addition of Congo red to the growth medium and the intracellular pools of PopB and PopC are very similar in the absence or presence of Congo red. Preliminary data suggest that Congo red stabilizes secreted proteins in the extracellular medium. PopB is a 173‐amino‐acid‐basic protein that contains a functional bipartite nuclear localization signal. PopC is a 1024‐amino‐acid protein that carries 22 tandem leucine‐rich repeats (LRR). The LRR domain of this protein forms a consensus that perfectly matches the predicted eukaryotic cytoplasmic LRR consensus. We propose that PopB and PopC may be translocated into plant cells via the Hrp pathway.

A switch in Ca2+ spiking signature is concomitant with endosymbiotic microbe entry into cortical root cells of Medicago truncatula.

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

Endoplasmic Microtubules Configure the Subapical Cytoplasm and Are Required for Fast Growth of Medicago truncatula Root Hairs

To investigate the configuration and function of microtubules (MTs) in tip-growing Medicago truncatularoot hairs, we used immunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similar results and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages. During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hair and remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional array throughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmic dense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, the only remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growing hairs with 1 μm oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes very short, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at a much larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense region remains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with 1 μm oryzalin. The role of EMTs in growing root hairs is discussed.

Nod Factors Alter the Microtubule Cytoskeleton in Medicago truncatula Root Hairs to Allow Root Hair Reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.

Nodulation Studies in the Model Legume Medicago truncatula: Advantages of Using the Constitutive EF1α Promoter and Limitations in Detecting Fluorescent Reporter Proteins in Nodule Tissues

The Cauliflower mosaic virus 35S promoter currently is being used in RNAi-based approaches for attenuating host gene expression during legume root nodule development and also for the expression of fluorescent reporters in nodule tissues. In this study, we have evaluated the expression of this promoter in the indeterminate nodules of the model plant Medicago truncatula. Our results clearly show that the 35S promoter is inactive in both the nodule meristem and in bacteroid-containing cells of the nodules. On the other hand, the Arabidopsis thaliana EF1alpha promoter was found to be strongly expressed both in the nodule meristem and in all nodule-invaded cells. Therefore, we conclude that the constitutive EF1alpha promoter is far superior for mRNAi or overexpression studies in nodule tissues compared with the commonly used 35S promoter. In addition, our experiments have revealed that the intensity of fluorescent markers such as green fluorescent protein is severely attenuated within invaded cells in the nitrogen-fixation zone of the nodule, most likely by fluorescence quenching. This phenomenon may hinder the use of these tools for live-cell imaging in nodule tissue.

The role of the plant cytoskeleton in the interaction between legumes and rhizobia

The study of the symbiotic interaction between rhizobia and legumes represents a major theme in plant biology. This interaction results in the formation of nodules, root organs in which the bacteria reduce atmospheric nitrogen into ammonia, which can subsequently be utilized by the plant. The execution of the different developmental stages observed during nodule ontogenesis involves many cellular processes with significant roles for the plant cytoskeleton. A challenging question in cell biology is how the cytoskeleton organizes itself into the dynamic arrays required for cell differentiation and functioning. Nodulation is, particularly, well qualified as an experimental system for cytoskeleton research because an early essential step of the plant/microbe interaction takes place in surface‐exposed root hairs, well suited for cell biological in vivo experimentation. Moreover, the changes in the organization of the cytoskeleton can be elicited by a well‐defined molecule, the Nod factor, or by bacterial inoculation, thus providing the researcher with the possibility of controlling the cytoskeletal changes in target cells. In addition, the well‐known cytology of the symbiotic interaction facilitates the correlation between the changes in the organization of the plant cytoskeleton with both histological and cellular changes. In this review, the current knowledge on the role of the plant cytoskeleton during nodulation is summarized, with emphasis on the interaction between Medicago truncatula and Sinorhizobium meliloti.

Microtubule dynamics during preprophase band formation and the role of endoplasmic microtubules during root hair elongation.

Plant cells possess a number of distinct microtubule cytoskeleton configurations that are successively being formed, remodeled and broken down during the life of a cell: the interphase cortical microtubules and those encaging the nucleus, the cortical preprophase band, the spindle and the phragmoplast. In order to understand whether and how dynamic instability of microtubules contributes to the transition from a cortical array to the preprophase band, we studied the dynamic instability of individual microtubules in BY2 cells expressing a GFP-microtubule binding domain (GFP-MBD) fusion protein. The step from cortical array to preprophase band was also studied at the level of the microtubule population dynamics. The results showed that microtubule dynamic instability is altered during preprophase band formation and that the process is biphasic. Interphase plant cells have not been reported to possess endoplasmic microtubules, except around the nucleus. However, using both a GFP-MBD fusion protein and immuno-cytochemistry after freeze fixation/ freeze substitution, we found endoplasmic microtubules in the sub-apical area of root hairs of the legume Medicago truncatula. Drug experiments showed that these microtubules are less stable than the cortical microtubules. Real-time 4D confocal microscopy revealed that the endoplasmic microtubules in the subapical region are very dynamic, constantly changing between phases of growth and shrinkage. Moreover, single microtubules were observed to grow from the sub-apical region into the very tip of the root hair. The endoplasmic sub-apical microtubules contribute to the organization of the cell, i.e. the cell architecture, which includes keeping the nucleus at a certain distance from the growing root hair tip. After depolymerization of the endoplasmic microtubules only, the distance between the growing root hair tip and the nucleus became larger, but the nucleus still followed the expanding tip, albeit at a larger distance than before depolymerization of the endoplasmic microtubules. Depolymerization of sub-apical endoplasmic microtubules, alone or along with the cortical microtubules, retarded, but did not stop, hair elongation. This novel microtubule array has not been reported for Arabidopsis root hairs. Further study should show whether it is specific for legumes and related to their interaction with Rhizobium bacteria. * Corresponding author. Tel.: +31-317-484329; fax: +31-317-485005. E-mail address: annemie.emons@pcb.dpw.wag-ur.nl (A.M.C. Emons). Cell Biology International 27 (2003) 295 Cell Biology International

Light microscopy of whole plant organs

Plants are ideal organisms for light microscopical studies of cellular mechanisms controlling cell organisation and cell functioning. However, most plant organs are not transparent to light which prevents high resolution imaging deep within plant tissues. Classically, access into plant organs is achieved by sectioning or whole‐mount tissue clearing. Until recently, the protocols for clearing destroyed the signal from fluorescent markers which prevented the imaging of the distribution of fluorescent proteins and the three‐dimensional reconstruction from optical slices of whole plant organs. From 2011, a number of protocols have been developed for whole brain and whole organism imaging for animal studies. Now, these protocols have been adapted for in‐depth imaging of whole plant organs. Here, I present an overview of clearing techniques of plant organs and highlight the latest developments of plant tissue clearing in combination with high resolution fluorescence microscopy.