Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity

Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.     

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.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

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.     

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.).     

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.     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Correlated behavior implicates stromules in increasing the interactive surface between plastids and ER tubules

Stromules are extended by plastids but the underlying basis for their extension and retraction had not been understood until recently. Our live-imaging aided observations on coincident plastid stromule branching and ER tubule dynamics open out new areas of investigation relating to these rapid subcellular interactions. This addendum provides a testable hypothesis on the formation of stromules, which argues against the need for new membrane incorporation and suggests that stromal extensions might result from a remodeling of the plastid envelope membrane in an ER aided manner.Key words: stromules, plastids, endoplasmic reticulum, fluorescent proteins, subcellular interactions, FNR-EGFP, RFP-ERThe extension and retraction of stromules (stroma-filled tubules) from both chlorophyll containing and achlorophyllous plastids is well established for diverse plant species.¹ Many different conditions such as increased subcellular redox stress,² symbiotic interactions,^3–5 elevated temperatures,⁶ viral infection⁷ and alterations in plastid size and density^8,9 have been associated with stromule formation. Stromules extended from different plastids have been observed as forming connecting bridges^10–13 through which an exchange of proteins has been demonstrated.^{10,11,14–16} The latter observations strongly suggested stromules as transient communication channels between plastids for exchanging metabolites.^10,11 The connectivity of plastids via stromules also led to the suggestion that plant cells might contain a plastidom, defined as a well-connected plastid-compartment.¹² However, as pointed out by Netasan and co-workers¹⁷ most stromules do not appear to interconnect plastids and thus the movement of macromolecules between plastids might not be their sole function. In concurrence with this viewpoint the observations of Gunning¹⁸ and Lütz and Engel¹⁹ suggest that stromules have a role in increasing plastid interactions with mitochondria and peroxisomes. Thus a more generalized and acceptable statement is that stromules serve to increase the plastid stromal surface area that is exposed to the subcellular environment.Notably, the largest membrane surface area within a cell is provided by the endoplasmic reticulum (ER). Transmission electron micrographs often suggest a close proximity between plastids and the ER^20,21 and the presence of a chloroplast envelope associated-ER has been demonstrated.²² However, studies aimed at uncovering possible dynamic interactions between stromules and the ER in living plant cells had not been carried out. Our recent work²³ investigated this possible relationship by simultaneously visualizing stromules (labeled using a ferredoxin NADP(H) oxidoreductase (FNR) transit peptide fused to enhanced GFP; hereafter referred to as FNR-EGFP) and the ER (highlighted using a chimeric red fluorescent protein (RFP) carrying a basic chitinase signal sequence and an ER retention sequence; referred to as RFP-ER). Our observations clearly identified the ER as providing the nearest set of membranes with which the plastid envelope can interact. The observations are most pertinent for the narrow cortical sleeve where the relatively large sized plastids (including chloroplasts) are pressed against the cell boundary (plasma membrane and comparatively rigid cell wall) on one side and a turgid vacuole on the inner side. This space is also traversed by the cortical ER mesh created by constantly reorganizing ER-tubules. As shown in Figure 1A the side of a plastid pressed against the plasma membrane is surrounded by a loose mesh of ER tubules while the side pressed close to the vacuolar membrane (Fig. 1B) is free of the ER cradle. The cortically located plastids display strong behavioral correlations between their stromules and the neighboring ER tubules (Fig. 1C–E). Notably stromules form triangular junctions that appear very similar to the 3-way junctions displayed by the cortical ER. The three-way junctions of the ER are cisternal locations from which tubules are extended to create ER polygons. Stromule branches are extended from the stromal triangles in a very similar manner along comparable angles. Interestingly the extension and retraction of stromules and their branches occurs in tandem with contiguous ER tubules. These observations on the coincident behavior of the two organelles indicate that stromule branching and dynamic behavior might rely upon the creation of multiple membrane contact sites (MCSs) with the ER. The MCSs could aid in stretching stromules along ER tubules while loss of contact between the two membranes could cause rapid stromule retraction. While the possibility of identifying MCSs on the stromule envelope is being explored further by us our initial observations evoke a long-standing but very relevant question. Where does the membrane for forming stromules come from?Open in a separate windowFigure 1Confocal laser scanning microscopy based imaging of living Nicotiana benthamiana cells co-expressing FNR-EGFP (labels stromules) and RFP-ER (labels ER), 3D isosurface rendering and a model based on the observations. (A) 3D iso-surface rendering of a chloroplast facing the plasma membrane side shows the plastid situated in a loose cradle of ER tubules. (B) The 3D rendered chloroplast ER reconstruction seen in (A) observed from the vacuolar side after rotation by 180° shows the chloroplast appears smooth and free of the ER mesh. (C–E) Sequential frames from a time-lapse movie depict a FNR-EGFP labeled stromule undergoing a branching event. Note that in (C) the stromule extending along an ER channel with possible contacts at several points along the stromule length. (D) depicts a stromal triangle (arrowhead) that forms a branch initial. (E) depicts a branched stromule with the branches aligned with the ER (size bar = 5 µm). (F) A plastid exhibiting a short, wide stroma-filled area after stromule retraction has occured. (G) The same plastid shown in (F) without the FNR-EGFP labeled stroma (arrowhead) provides an appreciation of the “mobile jacket” created by the stromal contents around the chlorophyll containing thylakoids (size bar = 5 µm). (H and I) A diagrammatic depiction based on our observations and pertinent literature. (H) The narrow cortical region between the vacuolar membrane and the cell boundary is shared by large plastids and a dynamic ER. A depiction of the loose stroma filled jacket (arrowhead) of a plastid along with contiguous ER. Note the relative positions of the vacuolar membrane and the plasma membrane. The plastid size cannot increase isotropically within the narrow confines of the cortex and thus stromule formation along ER tubules is favored (Arrow pointing direction of stromule extension along the ER). (I) Multiple contact sites might be created between the extending stromule and the neighboring ER tubules. The diagrammatic depiction emphasizes the remodeling of the loose plastid jacket for stromule extension along ER. The schematic does not depict the strong possibility that both organelles might share an F-actin based mechanism for their extension.Stromule extension visibly enlarges the area occupied by the stromal contents of a plastid. The general conclusion of stromules increasing the plastid surface is clearly validated. Indeed Hanson and coworkers¹ calculated that for a model plastid of 3 µm diameter extending a 0.2 µm diameter and 10 µm long stromule the stromule represents about 20% of the overall plastid envelope surface area. A point that remains unclear is whether stromule extension involves a net increase in plastidic membrane or merely involves a remodeling of the existing membrane into an elongated shape. In the first scenario stromule formation would involve the creation of fresh membrane vesicles and their incorporation into an existing bi-layered plastid coat. This would be followed by their dispersal into inner and outer plastid envelopes along with complete complements of protein import and export machinery. Currently there is no experimental evidence to support this scenario. Moreover, this viewpoint does not consider observations of stromule retraction at all. If new membranes have been added during stromule extension then what happens to them during the retraction stage? Further, live imaging clearly shows that the time involved in sporadic stromule extension, branching and retraction is in the order of seconds to minutes. Such short periods do not favor the viewpoint involving creation of new membrane and its incorporation into stromules.Interestingly, observations of plastids in living cells often suggest that they have a rather wobbly form than a tight compact shape. The unstable form suggests the presence of relaxed membranes enveloping the main plastid body (Fig. 1F and G). These loose fitting membranes have been described as a “mobile jacket”^1,24 whose presence and irregular protrusions can be clearly distinguished from long stromules using a shape index.⁶ Hanson and co-workers¹ speculated that the “mobile jacket” might be stretched out and could thus provide the extra membrane needed for stromule formation. The notion is supported by the work of Gunning, which shows stromules retracting into short amorphous protrusions.¹⁸ Our observations of short protrusions or “beaks”²³ being created sporadically on a relatively regular-appearing plastid surface support these ideas. According to our observations one or two of the beaks might develop into long stromules that would be extended along neighboring ER tubules (Fig. 1H and I).Moreover, in vitro experiments on sucrose filled liposomes and giant vesicles^25,26 are particularly interesting in this context. Plasmolytic shrinkage of the sucrose filled vesicles creates states in which excess membrane surrounds a small volume. Over time the membrane exhibits small protrusions that are very similar in shape to the protrusions formed by plastid. Taken together the above-mentioned observations support behavior that might be expected upon a reshaping of already existing membranes. Notably the membrane-remodeling scenario for the formation of a stromule does not involve an increase in the net membrane of a plastid but it does achieve an increase in the surface area over which plastid stromal contents can interact with their surroundings. The viewpoint is also able to explain stromule elasticity that is suggested by their rapid extension and retraction.The new observations and ensuing discussion suggesting stromules being formed by stretching of the plastid envelope and identifying the ER as an interactive membrane partner in the process lay down the foundation for a number of other questions. These include investigations relating to the role of stromules in metabolite import and export between the two organelles, understanding the relationship between stromule and the ER behavior in relation to the underlying actin cytoskeleton as well as assessing the specificity of myosin motors that might be involved in these dynamic processes.     

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.     

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N_1-107) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N_1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co-evolution.     

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.     

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.     

The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as-yet-unidentified avirulence factor possessed by the Japanese tobacco-avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector-triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence-related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid-resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco-virulent strain BK1002 contains mutations in ripB and expresses a C-terminal-truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine-tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB-induced resistance against R. solanacearum was abolished in Roq1-silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.     

Stromules: a characteristic cell-specific feature of plastid morphology

Stromules (stroma-filled tubules) are highly dynamic structures extending from the surface of all plastid types examined so far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts. Stromules are usually 0.35-0.85 microm in diameter and of variable length, from short beak-like projections to linear or branched structures up to 220 mum long. They are enclosed by the inner and outer plastid envelope membranes and enable the transfer of molecules as large as Rubisco (approximately 560 kDa) between interconnected plastids. Stromules occur in all cell types, but stromule morphology and the proportion of plastids with stromules vary from tissue to tissue and at different stages of plant development. In general, stromules are more abundant in tissues containing non-green plastids, and in cells containing smaller plastids. The primary function of stromules is still unresolved, although the presence of stromules markedly increases the plastid surface area, potentially increasing transport to and from the cytosol. Other functions of stromules, such as transfer of macromolecules between plastids and starch granule formation in cereal endosperm, may be restricted to particular tissues and cell types.     

Suppression of XopQ–XopX‐induced immune responses of rice by the type III effector XopG

Effectors that suppress effector‐triggered immunity (ETI) are an essential part of the arms race in the co‐evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ–XopX‐mediated immune responses. Both mutations individually affect the virulence‐promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.     

Trafficking of Proteins through Plastid Stromules

Stromules are thin projections from plastids that are generally longer and more abundant on non-green plastids than on chloroplasts. Occasionally stromules can be observed to connect two plastid bodies with one another. However, photobleaching of GFP-labeled plastids and stromules in 2000 demonstrated that plastids do not form a network like the endoplasmic reticulum, resulting in the proposal that stromules have major functions other than transfer of material from one plastid to another. The absence of a network was confirmed in 2012 with the use of a photoconvertible fluorescent protein, but the prior observations of movement of proteins between plastids were challenged. We review published evidence and provide new experiments that demonstrate trafficking of fluorescent protein between plastids as well as movement of proteins within stromules that emanate from a single plastid and discuss the possible function of stromules.Projections from chloroplasts have been reported sporadically in the literature for over a hundred years (reviewed in Gray et al., 2001; Kwok and Hanson, 2004a) and became established as genuine features of plastids when they were observed by the targeting of green fluorescent protein (GFP) to the stromal compartment (Köhler et al., 1997). This study showed that these projections sometimes appeared to connect discrete plastid bodies, and photobleaching experiments demonstrated flow of GFP from one plastid body to another. After GFP in one plastid body was bleached, fluorescence rapidly recovered as a result of flow from GFP from the unbleached plastids. By continuous bleaching of a stromule connecting two plastids, fluorescence was lost from both plastids. This led to the speculation that there could be an interplastid communication system (Köhler et al., 1997). In a follow-up study to test the degree of interplastid connectedness, the term “stromule” was coined to prevent confusion with other tubular structures in the cell (Köhler and Hanson, 2000). The existence of a stromule-based plastid network was ruled out by these experiments, but movement of protein through stromules was confirmed, and it was proposed that stromules might function to facilitate transport of substances in and out of the plastid by increasing surface area and by placing the plastid compartment in close proximity to other organelles or subcellular structures (Köhler and Hanson, 2000). A study by Schattat et al. (2012) confirmed the absence of a plastid network with the use of a photoconvertible fluorescent protein. These authors also describe photoconversion experiments that appear to contradict our prior work demonstrating flow of GFP between two plastid bodies connected by a stromule. Here, we confirm our prior fluorescence recovery after photobleaching (FRAP) results, showing that proteins can move through stromules between individual plastids, and we demonstrate that a red photoconverted protein can also move into a region where photoconversion has not occurred, provided that potentially damaging levels of light are not used during the photoconversion experiment. We review previous studies showing the lack of an interconnected plastid network and consider other functions for stromules, such as facilitating the transport of enzymes and metabolites to and from the plastid to the vicinity of other organelles or regions of the cell.     

Tight regulation of plant immune responses by combining promoter and suicide exon elements

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.     

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide‐binding leucine‐rich repeat (NLR) protein with a Toll‐like interleukin‐1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely‐related Nicotiana sylvestris and the distantly‐related beet plant (Beta vulgaris). Roq1 was found to co‐immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.     

Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics

Stromules are stroma-filled tubules extending from plastids whose rapid extension toward or retraction from other plastids has suggested a role in interplastidic communication and exchange of metabolites. Several studies point to sporadic dilations, kinks, and branches occurring along stromule length but have not elucidated the underlying basis for these occurrences. Similarly, although specific details on interacting partners have been missing, a consensus viewpoint suggests that stromules increase the interactive surface of a plastid with its cytoplasmic surroundings. Here, using live imaging, we show that the behavior of dynamic, pleomorphic stromules strongly coincides with that of cortical endoplasmic reticulum (ER) tubules. Covisualization of fluorescent protein-highlighted stromules and the ER in diverse cell types clearly suggests correlative dynamics of the two membrane-bound compartments. The extension and retraction, as well as directional changes in stromule branches occur in tandem with the behavior of neighboring ER tubules. Three-dimensional and four-dimensional volume rendering reveals that stromules that extend into cortical regions occupy channels between ER tubules possibly through multiple membrane contact sites. Our observations clearly depict coincidental stromule-ER behavior and suggest that either the neighboring ER tubules shape stromules directly or the behavior of both ER and stromules is simultaneously dictated by a shared cytoskeleton-based mechanism. These new observations strongly implicate the ER membrane in interactions with stromules and suggest that their interacting surfaces might serve as major conduits for bidirectional exchange of ions, lipids, and metabolites between the two organelles.     

The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death

Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events.