Effector XopQ-induced stromule formation in Nicotiana benthamiana depends on ETI signaling components ADR1 and NRG1

In Nicotiana benthamiana, the expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) triggers RECOGNITION OF XOPQ1 (ROQ1)-dependent effector-triggered immunity (ETI) responses accompanied by the accumulation of plastids around the nucleus and the formation of stromules. Both plastid clustering and stromules were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here, we utilized transient expression experiments to determine whether XopQ-triggered plastid reactions are a result of XopQ perception by the immune receptor ROQ1 or a consequence of XopQ virulence activity. We found that N. benthamiana mutants lacking ROQ1, ENHANCED DISEASE SUSCEPTIBILITY 1, or the helper NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT IMMUNE RECEPTORS (NLRs) N-REQUIRED GENE 1 (NRG1) and ACTIVATED DISEASE RESISTANCE GENE 1 (ADR1), fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the nrg1_adr1 double mutant. This analysis aligns XopQ-triggered stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response and that both NRG1 and ADR1 hNLRs play a role in this ETI response.

Genetic analysis aligns effector triggered immunity (ETI)-induced stromule formation with ETI signaling but not programmed cell death and questions stromule-guided perinuclear plastid clustering.     

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.     

The Xanthomonas effector XopL uncovers the role of microtubules in stromule extension and dynamics in Nicotiana benthamiana

Xanthomonas campestris pv. vesicatoria type III‐secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in Nicotiana benthamiana lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus. Further characterization of XopL revealed that the E3 ligase activity is essential for the two plastid phenotypes. In contrast to the XopL wild type, a mutant XopL lacking E3 ligase activity specifically localized to microtubules. Interestingly, mutant XopL‐labeled filaments frequently aligned with stromules, suggesting an important, yet unexplored, microtubule–stromule relationship. High time‐resolution movies confirmed that microtubules provide a scaffold for stromule movement and contribute to stromule shape. Taken together, this study has defined two populations of stromules: microtubule‐dependent stromules, which were found to move slower and persist longer, and microtubule‐independent stromules, which move faster and are transient. Our results provide the basis for a new model of stromule dynamics including interactions with both actin and microtubules.     

Chloroplast clustering around the nucleus is a general response to pathogen perception in Nicotiana benthamiana

It is increasingly clear that chloroplasts play a central role in plant stress responses. Upon activation of immune responses, chloroplasts are the source of multiple defensive signals, including reactive oxygen species (ROS). Intriguingly, it has been described that chloroplasts establish physical contact with the nucleus, through clustering around it and extending stromules, following activation of effector-triggered immunity (ETI). However, how prevalent this phenomenon is in plant–pathogen interactions, how its induction occurs, and what the underlying biological significance is are important questions that remain unanswered. Here, we describe that the chloroplast perinuclear clustering seems to be a general plant response upon perception of an invasion threat. Indeed, activation of pattern-triggered immunity, ETI, transient expression of the Rep protein from geminiviruses, or infection with viruses or bacteria all are capable of triggering this response in Nicotiana benthamiana. Interestingly, this response seems non-cell-autonomous, and exogenous treatment with H₂O₂ is sufficient to elicit this relocalization of chloroplasts, which appears to require accumulation of ROS. Taken together, our results indicate that chloroplasts cluster around the nucleus during plant–pathogen interactions, suggesting a fundamental role of this positioning in plant defence, and identify ROS as sufficient and possibly required for the onset of this response.     

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.     

Microfilaments and microtubules control the morphology and movement of non-green plastids and stromules in Nicotiana tabacum

Plastid stromules are stroma-filled tubular extensions of the plastid envelope membrane. These structures, which have been observed in a number of species, allow transfer of proteins between interconnected plastids. The dramatic shape of stromules and their dynamic movement within the cell provide an opportunity to study the control of morphology and motion of plastids. Using inhibitors of actin and tubulin, we found that both microfilaments and microtubules affect the shape and motility of non-green plastids. Actin and tubulin control plastid and stromule structure by independent mechanisms, while plastid movement is promoted by microfilaments but inhibited by microtubules. The presence or absence of stromules does not affect the motility of plastids. Photobleaching experiments indicate that actin and tubulin are not necessary for the bulk of green fluorescent protein (GFP) movement between plastids via stromules.     

TGD5 is required for normal morphogenesis of non-mesophyll plastids,but not mesophyll chloroplasts,in Arabidopsis

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.     

<Emphasis Type="Italic">In vivo</Emphasis>analysis of interactions between GFP-labeled microfilaments and plastid stromules

Background  

Plastid stromules are stroma-filled tubules that extend from the surface of plastids in higher plants and allow the exchange of protein molecules between plastids. These structures are highly dynamic; stromules change both their shape and position in the cytoplasm very rapidly. Previous studies with microfilament inhibitors indicated that stromule shape and movement are dependent on the actin cytoskeleton. To learn more about the nature of the interactions of stromules and the cytoskeleton, we imaged fluorescently-labeled microfilaments and plastids.     

Actin-based mechanisms for light-dependent intracellular positioning of nuclei and chloroplasts in Arabidopsis

The plant organelles, chloroplast and nucleus, change their position in response to light. In Arabidopsis thaliana leaf cells, chloroplasts and nuclei are distributed along the inner periclinal wall in darkness. In strong blue light, they become positioned along the anticlinal wall, while in weak blue light, only chloroplasts are accumulated along the inner and outer periclinal walls. Blue-light dependent positioning of both organelles is mediated by the blue-light receptor phototropin and controlled by the actin cytoskeleton. Interestingly, however, it seems that chloroplast movement requires short, fine actin filaments organized at the chloroplast edge, whereas nuclear movement does cytoplasmic, thick actin bundles intimately associated with the nucleus. Although there are many similarities between photo-relocation movements of chloroplasts and nuclei, plant cells appear to have evolved distinct mechanisms to regulate actin organization required for driving the movements of these organelles.Key words: actin, Arabidopsis, blue light, chloroplast positioning, phototropin, nuclear positioning     

Isolation and analysis of a stromule‐overproducing Arabidopsis mutant suggest the role of PARC6 in plastid morphology maintenance in the leaf epidermis

Stromules, or stroma‐filled tubules, are thin extensions of the plastid envelope membrane that are most frequently observed in undifferentiated or non‐mesophyll cells. The formation of stromules is developmentally regulated and responsive to biotic and abiotic stress; however, the physiological roles and molecular mechanisms of the stromule formation remain enigmatic. Accordingly, we attempted to obtain Arabidopsis thaliana mutants with aberrant stromule biogenesis in the leaf epidermis. Here, we characterize one of the obtained mutants. Plastids in the leaf epidermis of this mutant were giant and pleomorphic, typically having one or more constrictions that indicated arrested plastid division, and usually possessed one or more extremely long stromules, which indicated the deregulation of stromule formation. Genetic mapping, whole‐genome resequencing‐aided exome analysis, and gene complementation identified PARC6/CDP1/ARC6H, which encodes a vascular plant‐specific, chloroplast division site‐positioning factor, as the causal gene for the stromule phenotype. Yeast two‐hybrid assay and double mutant analysis also identified a possible interaction between PARC6 and MinD1, another known chloroplast division site‐positioning factor, during the morphogenesis of leaf epidermal plastids. To the best of our knowledge, PARC6 is the only known A. thaliana chloroplast division factor whose mutations more extensively affect the morphology of plastids in non‐mesophyll tissue than in mesophyll tissue. Therefore, the present study demonstrates that PARC6 plays a pivotal role in the morphology maintenance and stromule regulation of non‐mesophyll plastids.     

Visualisation of stromules in transgenic wheat expressing a plastid-targeted yellow fluorescent protein

Stromules are stroma-filled tubules that extend from the plastids in all multicellular plants examined to date. To facilitate the visualisation of stromules on different plastid types in various tissues of bread wheat (Triticum aestivum L.), a chimeric gene construct encoding enhanced yellow fluorescent protein (EYFP) targeted to plastids with the transit peptide of wheat granule-bound starch synthase I was introduced by Agrobacterium-mediated transformation. The gene construct was under the control of the rice Actin1 promoter, and EYFP fluorescence was detected in plastids in all cell types throughout the transgenic plants. Stromules were observed on all plastid types, although the stromule length and abundance varied markedly in different tissues. The longest stromules (up to 40 μm) were observed in epidermal cells of leaves, whereas only short beak-like stromules were observed on chloroplasts in mesophyll cells. Epidermal cells in leaves and roots contained the highest proportion of plastids with stromules, and stromules were also abundant on amyloplasts in the endosperm tissue of developing seeds. The general features of stromule morphology and distribution were similar to those shown previously for tobacco (Nicotiana tabacum L.) and arabidopsis (Arabidopsis thaliana (L.) Heynh.).     

Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging, and tip-shedding

Summary. Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations. Correspondence and reprints: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia.     

PEPRs spice up plant immunity

Some pattern recognition receptors (PRRs) in plants, such as PEPRs, sense endogenous, damage‐associated molecular patterns (DAMPs) that are released during pathogen infection. In this issue of The EMBO Journal, Yamada and colleagues show that genetic or pathogen‐induced depletion of Arabidopsis BAK1, a co‐receptor for multiple PRRs, primes immune activation through PEPRs. The work illustrates a link between pathogen‐induced perturbation of BAK1 and DAMP signaling.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary. In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance. Correspondence and reprints: Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Interaction of Actin and the Chloroplast Protein Import Apparatus

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.Actin is a ubiquitous protein of eukaryotic cells. Actin microfilaments are formed from polymerization of actin monomers and are a major component of the cytoskeleton. In plant cells, actin microfilaments are arranged in longitudinal arrays of thick actin bundles with randomly oriented thin actin filaments extending from the bundles (1). Chloroplasts are either aligned along the actin bundles or closely associated with the fine filaments and are surrounded by baskets of actin microfilaments (1, 2). A direct interaction of chloroplasts with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions (3). Chloroplast movement depends on cytosolic actin filaments and is stimulated by high light intensity (4). A chloroplast envelope protein involved in blue light-dependent chloroplast repositioning has been identified by the analysis of the Arabidopsis chup1 (chloroplast unusual positioning 1) mutant, which was unable to relocate its chloroplasts under high light stimulation (5). CHUP1 is a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane (6). CHUP1 interacts with actin and profilin, a modulator of actin polymerization, and it may play a regulatory role in actin polymerization during chloroplast photo-relocation (7).The interaction of amyloplasts with the actin cytoskeleton has been implicated in gravity perception and signal transduction. Several models for the role of the actin cytoskeleton have been proposed (8), but the nature of the interaction is not known. However, disruption of the actin cytoskeleton enhanced sedimentation of amyloplasts and promoted gravitropism (9, 10), and a role for myosin has been proposed on the basis of inhibitor experiments (11).The actin cytoskeleton and myosin have also been implicated in plastid stromule movement. Stromules (stroma-filled tubules) are highly dynamic tubular structures extending from the surface of all plastid types (12, 13). Stromules are delimited by the inner and outer plastid envelope membranes, which are closely associated (for a review, see Refs. 12 and 13). Experiments with inhibitors of microfilament- and microtubule-based movement suggested that stromules move along actin microfilaments powered by the ATPase activity of myosin motors (14). Physical connection between the envelope membranes seems likely to be required to provide a means of coordinating the movement of the inner envelope membrane with the microfilament-associated outer envelope membrane. There is evidence for direct connection of the inner and outer envelope membranes at contact sites, which support protein translocation through the protein import apparatus (15, 16). This apparatus consists of two membrane protein complexes that associate to allow translocation of nucleus-encoded proteins from the cytoplasm to the interior stromal compartment (for a review, see 17). The translocon at the outer envelope membrane of chloroplasts (TOC complex)² mediates the initial recognition of preproteins and their translocation across the outer membrane (18). The translocon at the inner envelope membrane of chloroplasts (TIC complex) physically associates with the TOC complex and provides the membrane translocation channel for the inner membrane. In addition, the TOC and TIC complexes interact with a set of molecular chaperones, which assist the transfer of imported proteins (19–21).With the aim of identifying components involved in the interaction of the chloroplast envelope with the actin cytoskeleton, we have used actin co-immunoprecipitation and co-sedimentation experiments with detergent-solubilized pea chloroplast envelope membranes. Components of the TOC-TIC protein import apparatus have been identified by mass spectrometry and Western blotting, and a direct interaction of Escherichia coli-expressed Toc159 with actin was demonstrated by co-sedimentation. This interaction may have a so far unrecognized physiological role in chloroplast protein import.     

Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of<Emphasis Type="Italic"> Vallisneria gigantea</Emphasis>

In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts.Preliminary results of part of the local irradiation study have already been reported in abstract form [N. Sakurai et al. (2002) J Photosci 9:326–328].     

Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris

Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.     

Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.     

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.