Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT

Subject Categories: Membranes & Trafficking, Microbiology, Virology & Host Pathogen Interaction, Structural Biology

We recently reported the first structures of the Plasmodium falciparum transporter PfFNT, both in the absence and presence of the inhibitor MMV007839 (Lyu et al, 2021). These structures indicated that PfFNT assembles as a pentamer. The bound MMV007839 was found in the middle of the elongated channel formed by each PfFNT protomer, adjacent to residue G107. MMV007839 exists in two tautomeric forms and can adopt either a cyclic hemiketal‐like structure or a linear vinylogous acid conformation (Fig ​(Fig3A).3A). Unfortunately, these two tautomeric forms could not be clearly distinguished based on the existing cryo‐EM data at 2.78 Å resolution. The bound MMV007839 inhibitor was reported as the cyclic hemiketal‐like form in the structure in Figs ​Figs3A3A and ​andF,F, and ​and4C,4C, Appendix Figs S10A and B, and S13 and in the online synopsis image.Open in a separate windowFigure 3Cryo‐EM structure of the PfFNT‐MMV007839 complex

1. Chemical structure of MMV007839. The compound can either be in cyclic hemiketal‐like or linear vinylogous acid tautomeric forms.
2. Cryo‐EM density map of pentameric PfFNT viewed from the parasite’s cytoplasm. Densities of the five bound MMV007839 within the pentamer are colored red. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple, and gray.
3. Ribbon diagram of the 2.18‐Å resolution structure of pentameric PfFNT‐MMV007839 viewed from the parasite’s cytoplasm. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple, and gray.
4. Ribbon diagram of pentameric PfFNT‐MMV007839 viewed from the extracellular side of the parasite. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple, and gray.
5. Ribbon diagram of pentameric PfFNT‐MMV007839 viewed from the parasite’s membrane plane. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple, and gray. Densities of the five bound MMV007839 are depicted as red meshes.
6. The MMV007839‐binding site of PfFNT. The bound MMV007839 is colored green. Density of the bound MMV007839 is depicted as black mesh. Residues involved in forming the inhibitor binding site are colored yellow. The hydrogen bonds are highlighted with black dotted lines.

Open in a separate windowFigure 4Structure of the central channel in the PfFNT‐MMV007839 protomer

• CA cartoon of the central channel formed within a PfFNT protomer. The channel contains one constriction site in this conformational state. Residues forming the constriction and the K35‐D103‐N108 and K177‐E229‐N234 triads are illustrated as sticks. Residues F94, I97, and L104, which form the first constriction site in the apo‐PfFNT structure, are also included in the figure.

Eric Beitz alerted us to the findings reported by his group that the linear vinylogous acid tautomer of MMV007839 constitutes the binding and inhibitory entity of PfFNT (Golldack et al, 2017).     

Experts in science communication: A shift from neutral encyclopedia to equal participant in dialogue

Even if the predominant model of science communication with the public is now based on dialogue, many experts still adhere to the outdated deficit model of informing the public. Subject Categories: Genetics, Gene Therapy & Genetic Disease, S&S: History & Philosophy of Science, S&S: Ethics

During the past decades, public communication of science has undergone profound changes: from policy‐driven to policy‐informing, from promoting science to interpreting science, and from dissemination to interaction (Burgess, 2014). These shifts in communication paradigms have an impact on what is expected from scientists who engage in public communication: they should be seen as fellow citizens rather than experts whose task is to increase scientific literacy of the lay public. Many scientists engage in science communication, because they see this as their responsibility toward society (Loroño‐Leturiondo & Davies, 2018). Yet, a significant proportion of researchers still “view public engagement as an activity of talking to rather than with the public” (Hamlyn et al, 2015). The highly criticized “deficit model” that sees the role of experts as educating the public to mitigate skepticism still persists (Simis et al, 2016; Suldovsky, 2016).Indeed, a survey we conducted among experts in training seems to corroborate the persistence of the deficit model even among younger scientists. Based on these results and our own experience with organizing public dialogues about human germline gene editing (Box 1), we discuss the implications of this outdated science communication model and an alternative model of public engagement, that aims to align science with the needs and values of the public.Box 1

The DNA‐dialogue project

The Dutch DNA‐dialogue project invited citizens to discuss and form opinions about human germline gene editing. During 2019 and 2020, this project organized twenty‐seven dialogues with professionals, such as embryologists and midwives, and various lay audiences. Different scenarios of a world in 2039 (https://www.rathenau.nl/en/making‐perfect‐lives/discussing‐modification‐heritable‐dna‐embryos) served as the starting point. Participants expressed their initial reactions to these scenarios with emotion‐cards and thereby explored the values they themselves and other participants deemed important as they elaborated further. Starting each dialogue in this way provides a context that enables everyone to participate in dialogue about complex topics such as human germline gene editing and demonstrates that scientific knowledge should not be a prerequisite to participate.An important example of “different” relevant knowledge surfaced during a dialogue with children between 8 and 12 years in the Sophia Children’s Hospital in Rotterdam (Fig 1). Most adults in the DNA‐dialogues accepted human germline gene modification for severe genetic diseases, as they wished the best possible care and outcome for their children. The children at Sophia, however, stated that they would find it terrible if their parents had altered something about them before they had been born; their parents would not even have known them. Some children went so far to say they would no longer be themselves without their genetic condition, and that their condition had also given them experiences they would rather not have missed.Open in a separate windowFigure 1 Children participating in a DNA‐dialogue meeting. Photographed by Levien Willemse.     

From the freezer to the clinic: Antifreeze proteins in the preservation of cells,tissues, and organs

Understanding the mechanisms by which natural anti‐freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

The first successful organ transplant in humans was performed in 1954 by Joseph Murray, who used a patient’s twin as a kidney donor. Murrays’ breakthrough paved the way for organ transplantation and the number of transplanted organs has grown ever since. For example, in 2017, a total of 139.024 solid organs—mostly kidney, liver, heart, lung, pancreas, and small bowel—were transplanted (Fig 1A). But this number only reflects 10% of the worldwide need; many patients still die of end‐stage organ failure while on a waiting list. The limited number of donor organs contributes only partially to this shortage. Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime. In fact, the percentage of donor organs that are unused is estimated to range from around 25% for kidneys and livers up to 70–80% for hearts and lungs (Giwa et al, 2017); Fig 1B).Open in a separate windowFigure 1Organ transplantation and preservability statusStatistics show a positive correlation between the duration of ex vivo preservation and the number of organ transplants. Number of solid organs transplanted in 2017 (A). Percentage of organs failed to be transplanted (B). Duration of solid organ ex vivo preservation in static cold storage (C). Sources: Data from the Global Observatory on Donation and Transplantation and (Parsons et al, 2014), (Guibert et al, 2011) and (Editorial: Buying time for transplants (2017))

Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime.

To address the shortage of donor organs and decrease the number of organs that go to waste, biobanks could efficiently store viable tissues and organs until transplantation. Yet, the current standard for ex vivo preservation of donor organs is static cold storage (4–8°C) which, depending on the organ, ensures viable conservation for only some hours; hearts are typically viable for a maximum of only 4 h (Fig 1C). In addition, this approach leads to hypothermic damage and to ischemia/reperfusion injury.Hence, there is an urgent need for strategies that prolong the viable preservation of donor organs. Two main strategies have emerged for cryopreservation and subzero storage, both of which cool tissues below the freezing point. While subzero storage just below 0°C may suffice for short‐term preservation, cryopreservation at −80°C or even lower temperatures is required for long‐term storage in biobanks. A proof‐of‐principle study already demonstrated that subzero preservation extended the preservation of rat hearts up to 24 h after collection (Amir et al, 2004); cryopreservation of whole hearts is currently not possible. The main reason is that lowering the temperature below the freezing point of water leads to ice formation, which causes cell damage and destroys tissues. One of the main challenges in biomedical research for organ transplantation is therefore finding non‐toxic and biocompatible antifreeze compounds that enable subzero storage and cryopreservation without causing tissue damage. An additional benefit is a larger time window to perform evaluation in terms of organ size and human leukocyte antigens matching and preparing the recipient patient to increase the chance of a successful transplantation.     

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1

Correction to: EMBO Reports (2019) 20: e47074. DOI 10.15252/embr.201847074 | Published online 6 May 2019The authors noticed that the control and disease labels had been inverted in their data analysis resulting in publication of incorrect data in Figure 1C. The corrected figure is displayed below. This change affects the conclusions as detailed below. The authors apologize for this error and any confusion it may have caused.In the legend of 1C, change from, “Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows increased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.Open in a separate windowFigure 1CCorrected Open in a separate windowFigure 1COriginal To, "Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows decreased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.In abstract, change from, “Here, we report increased IMP1 expression in patients with Crohn''s disease and ulcerative colitis”.To, “Here, we report increased IMP1 expression in adult patients with Crohn''s disease and ulcerative colitis”.In results, change from, “Consistent with these findings, analysis of published the Pediatric RISK Stratification Study (RISK) cohort of RNA‐sequencing data 38 from pediatric patients with Crohn''s disease (CD) patients revealed that IMP1 is upregulated significantly compared to control patients and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.To, “Contrary to our findings in colon tissue from adults, analysis of published RNA‐sequencing data from the Pediatric RISK Stratification Study (RISK) cohort of ileal tissue from children with Crohn’s disease (CD) 38 revealed that IMP1 is downregulated significantly compared to control patients in the RISK cohort and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.In discussion, change from, “Indeed, we report that IMP1 is upregulated in patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.To “Indeed, we report that IMP1 is upregulated in adult patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Bacterial metabolism and pathogenesis intimate intertwining: time for metabolic modelling to come into action

We take a snapshot of the recent understanding of bacterial metabolism and the bacterial‐host metabolic interplay during infection, and highlight key outcomes and challenges for the practical implementation of bacterial metabolic modelling computational tools in the pathogenesis field.

Once relegated to the supply of energy and biosynthetic precursors, it is now indubitable that metabolism mediates most of physiological processes. In the context of bacterial–host interactions where virulence is the outcome (commonly termed bacterial pathogenesis) metabolism expands far beyond its canonical role in bacterial proliferation. In addition to all sorts of recognized molecular determinants or virulence factors (toxins, flagella, translocated effectors, adhesins, invasins, etc.), bacterial pathogens are equipped with specific metabolic traits to circumvent immune defenses and antimicrobial killing, thus facilitating colonization and proliferation within their hosts. As the implementation of high‐throughput technologies elevates the pathogenesis field to the era of big data, it concurrently creates considerable challenges for our ability to interpret large data sets and identify factors that impact infectious processes. Metabolic modelling is emerging as a powerful tool allowing the integration and coherent organization of large data sets into the context of biological networks providing non‐intuitive insights on biological systems that experimental analysis alone cannot provide. Here, we take a snapshot of the recent understanding of bacterial metabolism and the bacterial–host metabolic interplay during infection, and highlight key outcomes and challenges for the practical implementation of bacterial metabolic modelling computational tools in the pathogenesis field (summarized in Fig. 1).Open in a separate windowFig. 1Genome‐scale metabolic network reconstructions for bacterial pathogenesis: it is time to leave a mark. Fast evolving advances in the genomics and metabolomics fields facilitate metabolic modelling of priority pathogens, of polymicrobial communities where key pathogens may have a starring role, and of host–pathogen systems. Metabolic reconstructions can yield significant benefits when combined with various layers of multi‐omics information as part of integration strategies, further enriched by the predictive potential of machine learning computational tools. Such integrative view will guide our experimental work to understand key metabolic traits in bacteria–bacteria or bacteria–host interactions where virulence is the outcome. More importantly, we foresee that such integrative view will contribute to pave the way for developing new diagnostic, treatment and surveillance procedures, seeking for their ultimate positive impact in the clinical management of bacterial infectious diseases.     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Conserved residues in the ankyrin domain of VAPYRIN indicate potential protein-protein interaction surfaces

Plant VAPYRINs are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINs of dicots, monocots and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.Key words: VAPYRIN, arbuscular mycorrhiza, petunia, symbiosis, glomus, ankyrin, major sperm protein, VAPPlants engage in mutualistic interactions such as root nodule symbiosis (RNS) with rhizobia and arbuscular mycorrhiza (AM) with Glomeromycotan fungi. These associations are referred to as endosymbioses because they involve transcellular passage through the epidermis and intracellular accommodation of the microbial partner within root cortical cells of the host.^1,2 Infection by AM fungi and rhizobia is actively promoted by the plant and requires the establishment of infection structures namely the prepenetration apparatus (PPA) in AM and a preinfection thread in RNS, respectively.^3–5 In both symbioses the intracellular microbial accommodation in epidermal and root cortical cells involves rebuilding of the cytoskeleton and of the entire membrane system.^6–8 Recently, intracellular accommodation of rhizobia and AM fungi, and in particular morphogenesis of the AM fungal feeding structures, the arbuscules, was shown to depend on the novel VAPYRIN protein.^9–11VAPYRINs are plant-specific proteins consisting of two protein-protein interaction domains, an N-terminal major sperm protein (MSP) domain and a C-terminal ankyrin (ANK) domain. MSP of C. elegans forms a cytoskeletal network required for the motility of the ameboidal sperm.¹² MSP domains also occur in VAP proteins that are involved in membrane fusion processes in various eukaryotes.¹³ The ANK domain, on the other hand, closely resembles animal ankyrins which serve to connect integral membrane proteins to elements of the spectrin cytoskeleton,¹⁴ thereby facilitating the assembly of functional membrane microdomains in diverse animal cells.¹⁵ Ankyrin repeats exhibit features of nano-springs, opening the possibility that ankyrin domains may be involved in mechanosensing.¹⁶ Based on these structural similarities, VAPYRIN may promote intracellular accommodation of endosymbionts by interacting with membranes and/or with the cytoskeleton. Indeed, VAPYRIN protein associates with small subcellular compartments in petunia and in Medicago truncatula.^9,10Ankyrin repeats typically consist of 33 amino acids, of which 30–40% are highly conserved across most taxa. These residues confer to the repeats their basic helix-turn-helix structure.¹⁷ Ankyrin domains often consist of arrays of several repeats that form a solenoid with a characteristic crescent shape.¹⁷ Besides the ankyrin-specific motiv-associated amino acids there is little conservation between the ankyrin domains of different proteins, or between the individual repeats of a given ankyrin domain,¹⁷ a feature that was also observed in petunia VAPYRIN (Fig. 1A).⁹ However, sequence comparison of VAPYRINs from eight dicots, three monocots and the moss Physcomitrella patens revealed a high degree of sequence conservation beyond the ankyrin-specific residues (Fig. 1B and Sup. Fig. S1). When the degree of conservation was determined for the individual ankyrin repeats among all the 12 species, it appeared that repeats 7, 9 and 10 exhibited particularly high conservation (Fig. 1C).Open in a separate windowFigure 1Sequence analysis and phylogeny of VAPYRIN from diverse plants. (A) Predicted amino acid sequence of the petunia VAPYRIN protein PAM1. The 11 repeats of the ankyrin domain are aligned, and the ankyrin consensus sequence is shown below the eleventh ankyrin repeat (line c). Conserved residues that are characteristic for ankyrin repeats (Mosavi et al. 2004)¹⁷ are depicted in bold face. (B) Unrooted phylogenetic tree representing the VAPYRINs of eight dicot species (Petunia hybrida, Solanum lycopersicon, Solanum tuberosum, Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula and Glycine max) three monocot species (Sorghum bicolor, Zea mays and Oryza sativa), and the moss Physcomitrella patens. (C) Degree of conservation of the individual ankyrin repeats of VAPYRIN. Schematic representation of the MSP domain as N-terminal barrel-shaped structure, and of the individual ankyrin repeats as pairs of alpha-helices. An additional loop occurring only in monocots (grass-loop) is inserted above repeat 4, and the deletion between repeat 7 and 8 is indicated (gap). This latter feature is common to all VAPYRIN proteins. The percentage of amino acid residues that are identical in at least 11 of the 12 VAPYRINS is given below the MSP domain and the eleven ankyrin repeats. The box highlights repeats 7–10 which contribute to the predicted binding site (compare with Figs. 3 and ​and44).Sequence comparison of the eleven repeats of all the twelve plant species revealed that the individual repeats clustered according to their position in the domain, rather than according to their origin (plant species) (Fig. 2). This shows that the repeats each are well conserved across species, but show little similarity among each other within a given VAPYRIN protein. The higher conservation of repeats 9 and 10 was reflected by the compact appearance of the respective branches, in which the monocot and moss sequences were nested closely with the dicot sequences, compared to other repeats, where the branches appeared fragmented between monocots and dicots, and where the P. patens sequence fell out of the branch as in the case of repeats 4–6 (Fig. 2). Taken together, this points to an old evolutionary origin of the entire ankyrin domain in lower land plants, with no subsequent rearrangement of ankyrin repeats.Open in a separate windowFigure 2Phylogenetic analysis of the individual ankyrin repeats of VAPYRIN. Phylogenetic representation of an alignment of all the 11 repeats of the 12 VAPYRINs compared in Figure 1B and C. The repeats cluster according to their position within the domain, rather than to their origin (plant species). Numbers indicate the position of the repeats within the domain (compare with Fig. 1C). P. patens repeats are highlighted (small circles) for clarity. The monocot repeat 4 sequences (boxed) are remote from the remaining repeat 4 sequences because of the grass loop (compare with Fig. 1C).Ankyrin domains function as protein-protein interaction domains,¹⁷ in which the residues on the surface are involved in the binding of their protein partners.¹⁴ The fact that repeats 9 and 10 exhibited particularly high levels of conservation across species from moss to angiosperms indicated that this region may contain functionally important residues. Within repeat 10, sixteen amino acid positions were identical in >90% of the analyzed species (Fig. 3A and grey bars). Nine of those represent residues that are characteristic for ankyrin repeats (red letters) and determine their typical 3D shape.¹⁷ These residues are considered ankyrin-specific, and are unlikely to be involved in a VAPYRIN-specific function. The remaining seven highly conserved residues in repeat 10, however, are VAPYRIN-specific, since they have been under positive selection, without being essential for the basic structure of the ankyrin repeat. Ankyrin-specific and VAPYRIN-specific residues where identified throughout the entire ankyrin domain (Sup. Fig. 1), and subsequently mapped on a 3-dimensional model of petunia VAPYRIN to reveal their position in the protein (Fig. 3B–G). The ankyrin-specific residues were found to be localized primarily to the interior of the ankyrin domain, with the characteristic glycines (brown) marking the turns between helices and loops (Fig. 3B, D and F, compare with A). In contrast, the VAPYRIN-specific residues were localized primarily on the surface of the ankyrin domain (Fig. 3C, E and G). A prominent clustering of VAPYRIN-specific residues was identified on the concave side of the crescent-shaped ankyrin domain comprising repeats 7–10 close to the gap (Figs. 3G and ​and44). This highly conserved VAPYRIN-specific region contains several negatively and positively charged residues (D, E and K, R, respectively) and aromatic residues (W, Y, F), which may together form a conserved binding site for an interacting protein.Open in a separate windowFigure 33D-Mapping of conserved positions within the ankyrin domain of VAPYRIN. (A) Conserved amino acid residues were evaluated for ankyrin repeat 10 of petunia VAPYRIN as an example. The degree of conservation between the 12 VAPYRINs analyzed in Figures 1B and ​and22 is depicted with grey bars. Average conservation between all the 132 ankyrin repeats of the 12 VAPYRIN sequences is shown with black bars. Residues that are conserved in all 132 repeats (red letters) define the ankyrin consensus sequence, which confers to the repeats their characteristic basic structure.¹⁷ Residues that are >90% conserved but are not part of the basic ankyrin sequence (highlighted with asterisks) are VAPYRIN-specific and may therefore have been conserved because of their specific function in VAPYRIN. Arrows indicate the characteristic antiparallel helices, the turns are marked by conserved glycine residues (underlined; compare with B, D and F). (B–G) 3D-models of the petunia VAPYRIN PAM1. Conserved amino acid residues were color-coded according to their physico-chemical properties (http://life.nthu.edu.tw/∼fmhsu/rasframe/SHAPELY.HTM) with minor modification (see below). In (B, D and F) the ankyrin-specific residues are highlighted (corresponding to the bold letters in Fig. 1A). In (C, E and G), the VAPYRIN-specific residues are highlighted. Note the patch of high conservation on the concave side of the crescent-shaped ankyrin domain between repeats 7–10 next to the gap. (B–E) represent respective side views of the ankyrin domain, (F and G) exhibit the concave inner side of the domain. Color code: Bright red: aspartic acid (D), glutamic acid (E); Yellow: cysteine (C); Blue: lysine (K), arginine (R); Orange: serine (S), threonine (T); Dark blue: phenylalanine (F), tyrosin (Y); Brown: glycine (G); Green: leucin (L), valine (V), isoleucin (I), alanine (A); Lilac: tryptophane (W); Purple: histidine (H); Pink: proline (P).Open in a separate windowFigure 4The highly conserved surface area in domain 8–10 of the ankyrin domain of petunia VAPYRIN. Close-up of the highly conserved region of petunia PAM1 as shown in Figure 3G. Amino acids were color-coded as in Figure 3 and their position in the amino acid sequence is indicated (compare with Sup. Fig. 1).In this context, it is interesting to note that human ankyrin R also contains a binding surface on the concave side of the D34 domain for the interaction with the CBD3 protein.¹⁴ Consistent with an essential function of the C-terminal third of the ankyrin domain, mutations that abolish this relatively short portion of VAPYRIN, have a strong phenotype, indicating that they may represent null alleles.⁹ Based on this collective evidence, we hypothesize that repeats 7–10 are involved in the formation of a protein complex that is essential for intracellular accommodation of rhizobia and AM fungi. Biochemical and genetic studies are now required to identify the binding partners of VAPYRINs, and to elucidate their role in plant endosymbioses.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

Focus Issue on Plant Cell Walls: Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins

Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its β-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure.Lignins are polymeric aromatic constituents of plant cell walls, constituting about 15% to 35% of the dry mass (Freudenberg and Neish, 1968; Adler, 1977). Unlike other natural polymers such as cellulose or proteins, which have labile linkages (glycosides and peptides) between their building units, lignins’ building units are combinatorially linked with strong ether and carbon-carbon bonds (Sarkanen and Ludwig, 1971; Harkin, 1973). It is difficult to completely degrade lignins. Lignins are traditionally considered to be dehydrogenative polymers derived from three monolignols, p-coumaryl alcohol 1h (which is typically minor), coniferyl alcohol 1g, and sinapyl alcohol 1s (Fig. 1; Sarkanen, 1971). They can vary greatly in their composition in terms of their plant and tissue origins (Campbell and Sederoff, 1996). This variability is probably determined and regulated by different activities and substrate specificities of the monolignol biosynthetic enzymes from different sources, and by the carefully controlled supply of monomers to the lignifying zone (Sederoff and Chang, 1991).Open in a separate windowFigure 1.The monolignols 1, and marker compounds 2 to 4 resulting from incorporation of novel monomer 15h into lignins: thioacidolysis monomeric marker 2, dimers 3, and DFRC dimeric markers 4.Recently there has been considerable interest in genetic modification of lignins with the goal of improving the utilization of lignocellulosics in various agricultural and industrial processes (Baucher et al., 2003; Boerjan et al., 2003a, 2003b). Studies on mutant and transgenic plants with altered monolignol biosynthesis have suggested that plants have a high level of metabolic plasticity in the formation of their lignins (Sederoff et al., 1999; Ralph et al., 2004). Lignins in angiosperm plants with depressed caffeic acid O-methyltransferase (COMT) were found to derive from significant amounts of 5-hydroxyconiferyl alcohol (5-OH-CA) monomers 15h (Fig. 1) substituting for the traditional monomer, sinapyl alcohol 1s (Marita et al., 2001; Ralph et al., 2001a, 2001b; Jouanin et al., 2004; Morreel et al., 2004b). NMR analysis of a ligqnin from COMT-deficient poplar (Populus spp.) has revealed that novel benzodioxane structures are formed through β-O-4 coupling of a monolignol with 5-hydroxyguaiacyl units (resulting from coupling of 5-OH-CA), followed by internal trapping of the resultant quinone methide by the phenolic 5-hydroxyl (Ralph et al., 2001a). When the lignin was subjected to thioacidolysis, a novel 5-hydroxyguaiacyl monomer 2 (Fig. 1) was found in addition to the normal guaiacyl and syringyl thioacidolysis monomers (Jouanin et al., 2000). Also, a new compound 3g (Fig. 1) was found in the dimeric products from thioacidolysis followed by Raney nickel desulfurization (Lapierre et al., 2001; Goujon et al., 2003).Further study with the lignin using the derivatization followed by reductive cleavage (DFRC) method also confirmed the existence of benzodioxane structures, with compounds 4 (Fig. 1) being identified following synthesis of the authentic parent compounds 9 (Fig. 2). However, no 5-hydroxyguaiacyl monomer could be detected in the DFRC products. These facts imply that the DFRC method leaves the benzodioxane structures fully intact, suggesting that the method might therefore be useful as an analytical tool for determining benzodioxane structures that are linked by β-O-4 ethers. Using a modified DFRC procedure, we report here on results that provide further evidence for the existence of benzodioxane structures in lignins from COMT-deficient plants, that 5-OH-CA is behaving as a rather ideal monolignol that can be integrated into plant lignins, and demonstrate the usefulness of the DFRC method for determining these benzodioxane structures.Open in a separate windowFigure 2.Synthesis of benzodioxane DFRC products 12 (see later in Fig. 6 for their structures). i, NaH, THF. ii, Pyrrolidine. iii, 1g or 1s, benzene/acetone (4/1, v/v). iv, DIBAL-H, toluene. v, Iodomethane-K₂CO₃, acetone. vi, Ac₂O pyridine.     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Novel Growth Regime of MDCK II Model Tissues on Soft Substrates

It is well established that MDCK II cells grow in circular colonies that densify until contact inhibition takes place. Here, we show that this behavior is only typical for colonies developing on hard substrates and report a new growth phase of MDCK II cells on soft gels. At the onset, the new phase is characterized by small, three-dimensional droplets of cells attached to the substrate. When the contact area between the agglomerate and the substrate becomes sufficiently large, a very dense monolayer nucleates in the center of the colony. This monolayer, surrounded by a belt of three-dimensionally packed cells, has a well-defined structure, independent of time and cluster size, as well as a density that is twice the steady-state density found on hard substrates. To release stress in such dense packing, extrusions of viable cells take place several days after seeding. The extruded cells create second-generation clusters, as evidenced by an archipelago of aggregates found in a vicinity of mother colonies, which points to a mechanically regulated migratory behavior.Studying the growth of cell colonies is an important step in the understanding of processes involving coordinated cell behavior such as tissue development, wound healing, and cancer progression. Apart from extremely challenging in vivo studies, artificial tissue models are proven to be very useful in determining the main physical factors that affect the cooperativity of cells, simply because the conditions of growth can be very well controlled. One of the most established cell types in this field of research is the Madin-Darby canine kidney epithelial cell (MDCK), originating from the kidney distal tube (1). A great advantage of this polarized epithelial cell line is that it retained the ability for contact inhibition (2), which makes it a perfect model system for studies of epithelial morphogenesis.Organization of MDCK cells in colonies have been studied in a number of circumstances. For example, it was shown that in three-dimensional soft Matrigel, MDCK cells form a spherical enclosure of a lumen that is enfolded by one layer of polarized cells with an apical membrane exposed to the lumen side (3). These structures can be altered by introducing the hepatocyte growth factor, which induces the formation of linear tubes (4). However, the best-studied regime of growth is performed on two-dimensional surfaces where MDCK II cells form sheets and exhibit contact inhibition. Consequently, the obtained monolayers are well characterized in context of development (5), mechanical properties (6), and obstructed cell migration (7–9).Surprisingly, in the context of mechanics, several studies of monolayer formation showed that different rigidities of polydimethylsiloxane gels (5) and polyacrylamide (PA) gels (9) do not influence the nature of monolayer formation nor the attainable steady-state density. This is supposedly due to long-range forces between cells transmitted by the underlying elastic substrate (9). These results were found to agree well with earlier works on bovine aortic endothelial cells (10) and vascular smooth muscle cells (11), both reporting a lack of sensitivity of monolayers to substrate elasticity. Yet, these results are in stark contrast with single-cell experiments (12–15) that show a clear response of cell morphology, focal adhesions, and cytoskeleton organization to substrate elasticity. Furthermore, sensitivity to the presence of growth factors that are dependent on the elasticity of the substrate in two (16) and three dimensions (4) makes this result even more astonishing. Therefore, we readdress the issue of sensitivity of tissues to the elasticity of the underlying substrate and show that sufficiently soft gels induce a clearly different tissue organization.We plated MDCK II cells on soft PA gels (Young’s modulus E = 0.6 ± 0.2 kPa), harder PA gels (E = 5, 11, 20, 34 kPa), and glass, all coated with Collagen-I. Gels were prepared following the procedure described in Rehfeldt et al. (17); rigidity and homogeneity of the gels was confirmed by bulk and microrheology (see the Supporting Material for comparison). Seeding of MDCK II cells involved a highly concentrated solution dropped in the middle of a hydrated gel or glass sample. For single-cell experiments, cells were dispersed over the entire dish. Samples were periodically fixed up to Day 12, stained for nuclei and actin, and imaged with an epifluorescence microscope. Details are described in the Supporting Material.On hard substrates and glass it was found previously that the area of small clusters expands exponentially until the movement of the edge cannot keep up with the proliferation in the bulk (5). Consequently, the bulk density increases toward the steady state, whereas the density of the edge remains low. At the same time, the colony size grows subexponentially (5). This is what we denote “the classical regime of growth”. Our experiments support these observations for substrates with E ≥ 5 kPa. Specifically, on glass, colonies start as small clusters of very low density of 700 ± 200 cells/mm² (Fig. 1, A and B), typically surrounded by a strong actin cable (Fig. 1, B and C). Interestingly, the spreading area of single cells (Fig. 1 A) on glass was found to be significantly larger, i.e., (2.0 ± 0.9) × 10⁻³ mm². After Day 4 (corresponding cluster area of 600 ± 100 mm²), the density in the center of the colony reached the steady state with 6,800 ± 500 cells/mm², whereas the mean density of the edge profile grew to 4,000 ± 500 cells/mm². This density was retained until Day 12 (cluster area 1800 ± 100 mm²), which is in agreement with previous work (9).Open in a separate windowFigure 1Early phase of cluster growth on hard substrates. (A) Well-spread single cells, and small clusters with a visible actin cable 6 h after seeding. (B) Within one day, clusters densify and merge, making small colonies. (C) Edge of clusters from panel B.In colonies grown on 0.6 kPa gels, however, we encounter a very different growth scenario. The average spreading area of single cells is (0.34 ± 0.3) × 10⁻³ mm², which is six times smaller than on glass substrates (Fig. 2 A). Clusters of only few cells show that cells have a preference for cell-cell contacts (a well-established flat contact zone can be seen at the cell-cell interface in Fig. 2 A) rather than for cell-substrate contacts (contact zone is diffusive and the shape of the cells appears curved). The same conclusion emerges from the fact that dropletlike agglomerates, resting on the substrate, form spontaneously (Fig. 2 A), and that attempts to seed one single cluster of 90,000 cells fail, resulting in a number of three-dimensional colonies (Fig. 2 A). When the contact area with the substrate exceeds 4.7 × 10⁻³ mm², a monolayer appears in the center of such colonies (Fig. 2 B). The colonies can merge, and if individual colonies are small, the collapse into a single domain is associated with the formation of transient irregular structures (Fig. 2 B). Ultimately, large elliptical colonies (average major/minor axis of e = 1.8 ± 0.6) with a smooth edge are formed (Fig. 2 C), unlike on hard substrates where circular clusters (e = 1.06 ± 0.06) with a ragged edge comprise the characteristic phenotype.Open in a separate windowFigure 2Early phase of cluster growth on soft substrates. (A) Twelve hours after seeding, single cells remain mostly round and small. They are found as individual, or within small, three-dimensional structures (top). The latter nucleate a monolayer in their center (bottom), if the contact area with the substrate exceeds ∼5 × 10⁻³ mm². (B) Irregularly-shaped clusters appear due to merging of smaller droplets. A stable monolayer surrounded by a three-dimensional belt of densely packed cells is clearly visible, even in larger structures. (C) All colonies are recorded on Day 4.Irrespective of cluster size, in the new regime of growth, the internal structure is built of two compartments (Fig. 2 B):

• 1.The first is the edge (0.019 ± 0.05-mm wide), a three-dimensional structure of densely packed cells. This belt is a signature of the new regime because on hard substrates the edge is strictly two-dimensional (Fig. 1 C).
• 2.The other is the centrally placed monolayer with a spatially constant density that is very weakly dependent on cluster size and age (Fig. 3). The mean monolayer density is 13,000 ± 2,000 cells/mm², which is an average over 130 clusters that are up to 12 days old and have a size in the range of 10⁻³ to 10 mm², each shown by a data point in Fig. 3. This density is twice the steady-state density of the bulk tissue in the classical regime of growth.Open in a separate windowFigure 3Monolayer densities in colonies grown on 0.6 kPa substrates, as a function of the cluster size and age. Each cluster is represented by a single data point signifying its mean monolayer density. (Black lines) Bulk and (red dashed lines) edge of steady-state densities from monolayers grown on glass substrates. Error bars are omitted for clarity, but are discussed in the Supporting Material.

Until Day 4, the monolayer is very homogeneous, showing a nearly hexagonal arrangement of cells. From Day 4, however, defects start to appear in the form of small holes (typical size of (0.3 ± 0.1) × 10⁻³ mm²). These could be attributed to the extrusions of viable cells, from either the belt or areas of increased local density in the monolayer (inset in Fig. 4). This suggests that extrusions serve to release stress built in the tissue, and, as a consequence, the overall density is decreased.Open in a separate windowFigure 4Cell nuclei within the mother colony and in the neighboring archipelago of second-generation clusters grown on 0.6 kPa gels at Day 12. (Inset; scale bar = 10 μm) Scar in the tissue, a result of a cell-extrusion event. (Main image; scale bar = 100 μm) From the image of cell nuclei (left), it is clear that there are no cells within the scar, whereas the image of actin (right) shows that the cytoplasm of the cells at the edge has closed the hole.Previous reports suggest that isolated MDCK cells undergo anoikis 8 h after losing contact with their neighbors (18). However, in this case, it appears that instead of dying, the extruded cells create new colonies, which can be seen as an archipelago surrounding the mother cluster (Fig. 4). The viability of off-cast cells is further evidenced by the appearance of single cells and second-generation colonies with sizes varying over five orders of magnitude, from Day 4 until the end of the experiment, Day 12. Importantly, no morphological differences were found in the first- and second-generation colonies.In conclusion, we show what we believe to be a novel phase of growth of MDCK model tissue on soft PA gels (E = 0.6 kPa) that, to our knowledge, despite previous similar efforts (9), has not been observed before. This finding is especially interesting in the context of elasticity of real kidneys, for which a Young’s modulus has been found to be between 0.05 and 5 kPa (19,20). This coincides with the elasticity of substrates studied herein, and opens the possibility that the newly found phase of growth has a particular biological relevance. Likewise, the ability to extrude viable cells may point to a new migratory pathway regulated mechanically by the stresses in the tissue, the implication of which we hope to investigate in the future.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.