53 results on '"Moeder W"'
Search Results
2. First report of the identification of a ‘ Candidatus Phytoplasma pruni‘‐related strain in Trillium species in Canada
- Author
-
Arocha‐Rosete, Y., primary, Morales‐Lizcano, N.P., additional, Hasan, A., additional, Yoshioka, K., additional, Moeder, W., additional, Michelutti, R., additional, Satta, E., additional, Bertaccini, A., additional, and Scott, J., additional
- Published
- 2016
- Full Text
- View/download PDF
3. Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco
- Author
-
Chamnongpol, S., Willekens, H., Moeder, W., Langebartels, C., Sandermann, H., Van Montagu, Marc, Inzé, Dirk, Laboratoire de biologie cellulaire et moléculaire, and Institut National de la Recherche Agronomique (INRA)
- Subjects
[SDV]Life Sciences [q-bio] ,ComputingMilieux_MISCELLANEOUS ,EXPRESSION GENETIQUE - Abstract
International audience
- Published
- 1998
4. First report of the identification of a ‘ Candidatus Phytoplasma pruni‘‐related strain in Trillium species in Canada
- Author
-
R. Michelutti, N.P. Morales-Lizcano, James A. Scott, E. Satta, Assunta Bertaccini, Wolfgang Moeder, Yaima Arocha-Rosete, Ahmed R. Hasan, Keiko Yoshioka, Arocha-Rosete, Y., Morales-Lizcano, N.P., Hasan, A., Yoshioka, K., Moeder, W., Michelutti, R., Satta, E., Bertaccini, A., and Scott, J.
- Subjects
0106 biological sciences ,0301 basic medicine ,biology ,Perennial plant ,Health, Toxicology and Mutagenesis ,food and beverages ,Plant Science ,030108 mycology & parasitology ,biology.organism_classification ,01 natural sciences ,Trillium ,03 medical and health sciences ,Melanthiaceae ,Genus ,Botany ,Temperate climate ,Identification (biology) ,Candidatus Phytoplasma pruni ,Medicinal plants ,Phytoplasmas, Trillium, Canada, 'Candidatus Phytoplasma pruni' ,Agronomy and Crop Science ,010606 plant biology & botany - Abstract
Trillium (family Melanthiaceae) is a genus of perennial flowering plants native to temperate regions of North America and Asia, and traditionally used as medicinal plants (Case, 2000). There are 49 species within the genus Trillium amongst which the white trillium, T. grandiflorum serves as the emblem and official flower of the Canadian province of Ontario. Other species include T. erectum, also known as red trillium. Many of these plants are adapted to conditions found in suburban or rural gardens. Compared with symptomless plants (Fig. 1), plants of T. grandiflorum and T. erectum with symptoms of virescence typical of phytoplasma infection (Figs. 2-3) were observed growing as weeds on a private property in the Sparta area of London, Ontario in May 2016 Plants were removed by the owner, placed in plastic bags and transported to the laboratory in ice coolers. Leaves, petioles and flowers were excised from eight symptom-bearing and two symptomless plants of each species. Total DNA was extracted and used as a template for a nested PCR assay with universal primers that target the phytoplasma 16S rRNA gene, R16mF2/R1 for the first PCR reaction, and R16F2n/R2 for the nested reaction (Gundersen & Lee, 1996). All samples from symptom-bearing plants yielded R16F2n/R2 amplicons. No PCR amplicons were obtained from the symptomless plants. One R16F2n/R2 amplicon from T. grandiflorum and three from T. erectum were purified (EZNA Cycle Pure Kit, Omega Bio-Tek, USA), cloned (pGEM-T Easy Vector, Promega) and sequenced (University of Toronto, Canada). Sequences were compared with those of phytoplasmas in NCBI. Sequences from T. grandiflorum (Genbank Accession No. KX470428) and T. erectum (KX470427, KX470429 and KX470430) shared over 99.7% sequence identity and were 99% similar to those of phytoplasma members of the group 16SrIII (Western X-disease). Phylogenetic analysis (using MEGA v4.0) of the 16S rDNA sequence (Fig. 4) supported the grouping of the phytoplasma isolated from both T. grandiflorum and T. erectum plants as a 'Candidatus Phytoplasma pruni'-related strain, with 99% sequence identity o the reference strain for the species (Milkweed yellows phytoplasma, 16SrIII-F, HQ589200). No phytoplasmas have been reported from Trillium plants in Canada. The X-disease (group 16SrIII) was first reported in 1941 in Ontario in the Niagara peninsula (Stevens & Stevens, 1947). In 1971, peach X-disease was detected in Essex County (Dhanvantari & Kappel, 1978) affecting more than 50% of peach orchards. In North America, phytoplasmas of the 16SrIII group are associated with several diseases in cherry and peach (Scott & Zimmerman, 2001). The presence of a 'Ca. P. pruni'-related strain in Trillium not only poses a threat for T. grandiflorum as a protected species, and other Trillium species in Ontario, but also for other plant species grown nearby. To our knowledge, this is the first detection of a phytoplasma in Trillium species in Canada and worldwide.
- Published
- 2016
5. Colour-analyzer: a new dual colour model-based imaging tool to quantify plant disease.
- Author
-
Loranger MEW, Yim W, Accomazzi V, Morales-Lizcano N, Moeder W, and Yoshioka K
- Abstract
Background: Despite major efforts over the last decades, the rising demands of the growing global population makes it of paramount importance to increase crop yields and reduce losses caused by plant pathogens. One way to tackle this is to screen novel resistant genotypes and immunity-inducing agents, which must be conducted in a high-throughput manner., Results: Colour-analyzer is a free web-based tool that can be used to rapidly measure the formation of lesions on leaves. Pixel colour values are often used to distinguish infected from healthy tissues. Some programs employ colour models, such as RGB, HSV or L*a*b*. Colour-analyzer uses two colour models, utilizing both HSV (Hue, Saturation, Value) and L*a*b* values. We found that the a* b* values of the L*a*b* colour model provided the clearest distinction between infected and healthy tissue, while the H and S channels were best to distinguish the leaf area from the background., Conclusion: By combining the a* and b* channels to determine the lesion area, while using the H and S channels to determine the leaf area, Colour-analyzer provides highly accurate information on the size of the lesion as well as the percentage of infected tissue in a high throughput manner and can accelerate the plant immunity research field., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
6. Editorial: Ca 2+ signalling in plant biotic interactions.
- Author
-
DeFalco TA, Moeder W, and Yoshioka K
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2023
- Full Text
- View/download PDF
7. A tale of many families: calcium channels in plant immunity.
- Author
-
Xu G, Moeder W, Yoshioka K, and Shan L
- Subjects
- Calcium Signaling, Cytoplasm metabolism, Cytosol metabolism, Humans, Plant Immunity genetics, Plants genetics, Plants metabolism, Calcium metabolism, Calcium Channels genetics, Calcium Channels metabolism
- Abstract
Plants launch a concerted immune response to dampen potential infections upon sensing microbial pathogen and insect invasions. The transient and rapid elevation of the cytosolic calcium concentration [Ca2+]cyt is among the essential early cellular responses in plant immunity. The free Ca2+ concentration in the apoplast is far higher than that in the resting cytoplasm. Thus, the precise regulation of calcium channel activities upon infection is the key for an immediate and dynamic Ca2+ influx to trigger downstream signaling. Specific Ca2+ signatures in different branches of the plant immune system vary in timing, amplitude, duration, kinetics, and sources of Ca2+. Recent breakthroughs in the studies of diverse groups of classical calcium channels highlight the instrumental role of Ca2+ homeostasis in plant immunity and cell survival. Additionally, the identification of some immune receptors as noncanonical Ca2+-permeable channels opens a new view of how immune receptors initiate cell death and signaling. This review aims to provide an overview of different Ca2+-conducting channels in plant immunity and highlight their molecular and genetic mode-of-actions in facilitating immune signaling. We also discuss the regulatory mechanisms that control the stability and activity of these channels., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2022
- Full Text
- View/download PDF
8. Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots.
- Author
-
Wang R, Himschoot E, Grenzi M, Chen J, Safi A, Krebs M, Schumacher K, Nowack MK, Moeder W, Yoshioka K, Van Damme D, De Smet I, Geelen D, Beeckman T, Friml J, Costa A, and Vanneste S
- Subjects
- Calcium metabolism, Indoleacetic Acids metabolism, Plant Growth Regulators metabolism, Plant Roots metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism
- Abstract
Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2022
- Full Text
- View/download PDF
9. Arabidopsis thaliana CYCLIC NUCLEOTIDE-GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis.
- Author
-
Wang L, Ning Y, Sun J, Wilkins KA, Matthus E, McNelly RE, Dark A, Rubio L, Moeder W, Yoshioka K, Véry AA, Stacey G, Leblanc-Fournier N, Legué V, Moulia B, and Davies JM
- Subjects
- Adenosine Triphosphate metabolism, Calcium metabolism, Cyclic Nucleotide-Gated Cation Channels genetics, Cyclic Nucleotide-Gated Cation Channels metabolism, Cyclic Nucleotide-Gated Cation Channels pharmacology, Epidermal Cells, Epidermis metabolism, Nucleotides, Cyclic metabolism, Nucleotides, Cyclic pharmacology, Signal Transduction, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca
2+ ]cyt ) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin). Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co-subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP-induced [Ca2+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)- Published
- 2022
- Full Text
- View/download PDF
10. CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling.
- Author
-
Chakraborty S, Toyota M, Moeder W, Chin K, Fortuna A, Champigny M, Vanneste S, Gilroy S, Beeckman T, Nambara E, and Yoshioka K
- Subjects
- Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Cyclic Nucleotide-Gated Cation Channels genetics, Cyclic Nucleotide-Gated Cation Channels physiology, Homeostasis, Indoleacetic Acids metabolism, Plant Growth Regulators physiology, Signal Transduction
- Abstract
Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca2+-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca2+ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca2+ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca2+ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS-YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca2+ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2021
- Full Text
- View/download PDF
11. Multiple phosphorylation events of the mitochondrial membrane protein TTM1 regulate cell death during senescence.
- Author
-
Karia P, Yoshioka K, and Moeder W
- Subjects
- Abscisic Acid metabolism, Abscisic Acid pharmacology, Acid Anhydride Hydrolases genetics, Arabidopsis drug effects, Arabidopsis Proteins genetics, Cell Death, Darkness, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Mitogen-Activated Protein Kinase Kinases metabolism, Phosphorylation, Plant Leaves drug effects, Plant Leaves metabolism, Plants, Genetically Modified, Serine metabolism, Acid Anhydride Hydrolases metabolism, Arabidopsis cytology, Arabidopsis physiology, Arabidopsis Proteins metabolism, Plant Senescence physiology
- Abstract
The role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein (MAP) kinases regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (Ser10, Ser437, and Ser490). Ser437, which is phosphorylated upon perception of senescence cues such as ABA and prolonged darkness, is phosphorylated by the MAP kinases MPK3 and MPK4, and Ser437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), also phosphorylate Ser10 and Ser490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-associated cell death, revealing a novel link between mitochondria and PCD., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2021
- Full Text
- View/download PDF
12. Impact of Maternal Intrapartum Antibiotics, and Caesarean Section with and without Labour on Bifidobacterium and Other Infant Gut Microbiota.
- Author
-
Chen YY, Zhao X, Moeder W, Tun HM, Simons E, Mandhane PJ, Moraes TJ, Turvey SE, Subbarao P, Scott JA, and Kozyrskyj AL
- Abstract
Background and Aims: Few studies consider the joint effect of multiple factors related to birth, delivery mode, intrapartum antibiotic prophylaxis and the onset of labour, on the abundance of Bifidobacterium and the quantity of this genus and its species Bifidobacterium longum subsp. infantis in the infant gut microbiota. We implemented such a study., Methods: Among 1654 Canadian full-term infants, the gut microbiota of faecal samples collected at 3 months were profiled by 16S rRNA sequencing; the genus Bifidobacterium and Bifidobacterium longum subsp. infantis were quantified by qPCR. Associations between Bifidobacterium and other gut microbiota were examined by Spearman's rank correlation., Results: Following vaginal birth, maternal IAP exposure was associated with reduced absolute quantities of bifidobacteria among vaginally delivered infants (6.80 vs. 7.14 log
10 (gene-copies/g faeces), p < 0.05), as well as their lowered abundance relative to other gut microbiota. IAP differences in infant gut bifidobacterial quantity were independent of maternal pre-pregnancy body-mass-index (BMI), and remarkably, they were limited to breastfed infants. Pre-pregnancy BMI adjustment revealed negative associations between absolute quantities of bifidobacteria and CS with or without labour in non-breastfed infants, and CS with labour in exclusively breastfed infants. Significant correlations between Bifidobacterium abundance and other microbial taxa were observed., Conclusions: This study documented the impact of the birth mode and feeding status on the abundance of gut Bifidobacterium , and pointed to the important ecological role of the genus Bifidobacterium in gut microbiota due to its strong interaction with other gut microbiota in early infancy.- Published
- 2021
- Full Text
- View/download PDF
13. Calcium channel in plants helps shut the door on intruders.
- Author
-
Yoshioka K and Moeder W
- Subjects
- Plants, Potassium Channels, Calcium, Calcium Channels
- Published
- 2020
- Full Text
- View/download PDF
14. Plant Cyclic Nucleotide-Gated Channels: New Insights on Their Functions and Regulation.
- Author
-
Dietrich P, Moeder W, and Yoshioka K
- Subjects
- Calcium Signaling genetics, Calcium Signaling physiology, Cyclic GMP metabolism, Cyclic Nucleotide-Gated Cation Channels genetics, HEK293 Cells, Humans, Neuronal Calcium-Sensor Proteins genetics, Neuronal Calcium-Sensor Proteins metabolism, Nucleotides, Cyclic metabolism, Phosphorylation genetics, Phosphorylation physiology, Cyclic Nucleotide-Gated Cation Channels metabolism
- Published
- 2020
- Full Text
- View/download PDF
15. The Receptor Kinases BAK1/SERK4 Regulate Ca 2+ Channel-Mediated Cellular Homeostasis for Cell Death Containment.
- Author
-
Yu X, Xu G, Li B, de Souza Vespoli L, Liu H, Moeder W, Chen S, de Oliveira MVV, Ariádina de Souza S, Shao W, Rodrigues B, Ma Y, Chhajed S, Xue S, Berkowitz GA, Yoshioka K, He P, and Shan L
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins physiology, Cell Death genetics, Cyclic Nucleotide-Gated Cation Channels physiology, Gene Expression Regulation, Plant genetics, Homeostasis, Phosphorylation, Plant Cells metabolism, Protein Kinases physiology, Protein Serine-Threonine Kinases physiology, Signal Transduction, Arabidopsis Proteins metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism, Protein Kinases metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca
2+ -permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca2+ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
16. A host-pathogen interactome uncovers phytopathogenic strategies to manipulate plant ABA responses.
- Author
-
Cao FY, Khan M, Taniguchi M, Mirmiran A, Moeder W, Lumba S, Yoshioka K, and Desveaux D
- Subjects
- Abscisic Acid metabolism, Arabidopsis genetics, Arabidopsis microbiology, Arabidopsis Proteins genetics, Bacterial Proteins genetics, Gene Expression Regulation drug effects, Host-Pathogen Interactions drug effects, Host-Pathogen Interactions genetics, Plant Diseases genetics, Plant Diseases microbiology, Plant Growth Regulators metabolism, Plant Growth Regulators pharmacology, Protein Interaction Maps genetics, Pseudomonas syringae genetics, Pseudomonas syringae pathogenicity, Virulence genetics, Abscisic Acid pharmacology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Bacterial Proteins metabolism, Protein Interaction Maps drug effects, Pseudomonas syringae metabolism
- Abstract
The phytopathogen Pseudomonas syringae delivers into host cells type III secreted effectors (T3SEs) that promote virulence. One virulence mechanism employed by T3SEs is to target hormone signaling pathways to perturb hormone homeostasis. The phytohormone abscisic acid (ABA) influences interactions between various phytopathogens and their plant hosts, and has been shown to be a target of P. syringae T3SEs. In order to provide insight into how T3SEs manipulate ABA responses, we generated an ABA-T3SE interactome network (ATIN) between P. syringae T3SEs and Arabidopsis proteins encoded by ABA-regulated genes. ATIN consists of 476 yeast-two-hybrid interactions between 97 Arabidopsis ABA-regulated proteins and 56 T3SEs from four pathovars of P. syringae. We demonstrate that T3SE interacting proteins are significantly enriched for proteins associated with transcription. In particular, the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors is highly represented. We show that ERF105 and ERF8 displayed a role in defense against P. syringae, supporting our overall observation that T3SEs of ATIN converge on proteins that influence plant immunity. In addition, we demonstrate that T3SEs that interact with a large number of ABA-regulated proteins can influence ABA responses. One of these T3SEs, HopF3
Pph6 , inhibits the function of ERF8, which influences both ABA-responses and plant immunity. These results provide a potential mechanism for how HopF3Pph6 manipulates ABA-responses to promote P. syringae virulence, and also demonstrate the utility of ATIN as a resource to study the ABA-T3SE interface., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)- Published
- 2019
- Full Text
- View/download PDF
17. Ca 2+ to the rescue - Ca 2+ channels and signaling in plant immunity.
- Author
-
Moeder W, Phan V, and Yoshioka K
- Subjects
- Calcium physiology, Plants immunology, Plants metabolism, Calcium metabolism, Calcium Channels metabolism, Plant Immunity physiology, Signal Transduction
- Abstract
Ca
2+ is a universal second messenger in many signaling pathways in all eukaryotes including plants. Transient changes in [Ca2+ ]cyt are rapidly generated upon a diverse range of stimuli such as drought, heat, wounding, and biotic stresses (infection by pathogenic and symbiotic microorganisms), as well as developmental cues. It has been known for a while that [Ca2+ ]cyt transient signals play crucial roles to activate plant immunity and recently significant progresses have been made in this research field. However the identity and regulation of ion channels that are involved in defense related Ca2+ signals are still enigmatic. Members of two ligand gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) have been implicated in immune responses; nevertheless more precise data to understand their direct involvement in the creation of Ca2+ signals during immune responses is necessary. Furthermore, the study of other ion channel groups is also required to understand the whole picture of the intra- and inter-cellular Ca2+ signalling network. In this review we summarize Ca2+ signals in plant immunity from an ion channel point of view and discuss future challenges in this exciting research field., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
18. Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity.
- Author
-
Cao FY, DeFalco TA, Moeder W, Li B, Gong Y, Liu XM, Taniguchi M, Lumba S, Toh S, Shan L, Ellis B, Desveaux D, and Yoshioka K
- Subjects
- Amino Acid Motifs, Arabidopsis cytology, Arabidopsis Proteins genetics, Arabidopsis Proteins immunology, Cell Death, Gene Expression Regulation, Plant, Mitogen-Activated Protein Kinases genetics, Mitogen-Activated Protein Kinases metabolism, Mutation, Phosphorylation, Plant Diseases, Plants, Genetically Modified, Pseudomonas syringae pathogenicity, Repressor Proteins genetics, Repressor Proteins immunology, Salicylic Acid metabolism, Serine genetics, Signal Transduction, Nicotiana genetics, Abscisic Acid metabolism, Arabidopsis physiology, Arabidopsis Proteins metabolism, Plant Immunity physiology, Repressor Proteins metabolism
- Abstract
Background: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses., Results: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively., Conclusions: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.
- Published
- 2018
- Full Text
- View/download PDF
19. CNGCs break through-A rice cyclic nucleotide-gated channel paves the way for pollen tube growth.
- Author
-
Moeder W and Yoshioka K
- Subjects
- Calcium metabolism, Cyclic Nucleotide-Gated Cation Channels genetics, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Models, Biological, Oryza genetics, Oryza growth & development, Plant Proteins genetics, Pollen Tube genetics, Pollen Tube growth & development, Pollination genetics, Seeds genetics, Seeds growth & development, Seeds metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism, Oryza metabolism, Plant Proteins metabolism, Pollen Tube metabolism
- Published
- 2017
- Full Text
- View/download PDF
20. Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.
- Author
-
Ung H, Karia P, Ebine K, Ueda T, Yoshioka K, and Moeder W
- Subjects
- Acid Anhydride Hydrolases genetics, Arabidopsis genetics, Arabidopsis physiology, Arabidopsis radiation effects, Arabidopsis Proteins genetics, Cell Death, Darkness, Gene Knockout Techniques, Genes, Reporter, Mitochondria enzymology, Mutation, Organ Specificity, Phenotype, Plant Leaves enzymology, Plant Leaves genetics, Plant Leaves physiology, Plant Leaves radiation effects, Polyphosphates metabolism, Protein Domains, Pyrophosphatases genetics, Nicotiana enzymology, Nicotiana genetics, Nicotiana physiology, Acid Anhydride Hydrolases metabolism, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Pyrophosphatases metabolism
- Abstract
The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis ( Arabidopsis thaliana ) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins., (© 2017 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2017
- Full Text
- View/download PDF
21. Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species.
- Author
-
DeFalco TA, Toyota M, Phan V, Karia P, Moeder W, Gilroy S, and Yoshioka K
- Subjects
- Arabidopsis genetics, Calcium metabolism, Cold Temperature, Gene Expression, Gene Silencing, Pathogen-Associated Molecular Pattern Molecules metabolism, Plant Leaves physiology, Plant Proteins genetics, Plants, Genetically Modified, Stress, Physiological, Nicotiana cytology, Nicotiana genetics, Calcium Signaling, Plant Proteins metabolism, Nicotiana physiology
- Abstract
Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2017
- Full Text
- View/download PDF
22. Calmodulin as a Ca2+-Sensing Subunit of Arabidopsis Cyclic Nucleotide-Gated Channel Complexes.
- Author
-
Fischer C, DeFalco TA, Karia P, Snedden WA, Moeder W, Yoshioka K, and Dietrich P
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Gene Expression Regulation, Plant, Protein Binding, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Calcium metabolism, Calcium Signaling, Calmodulin metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism
- Abstract
Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and the spatial and temporal patterns of Ca2+ concentration changes are determined by feedback and feed-forward regulation of the involved transport proteins. Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin (CaM). CNGCs interact with CaMs via diverse CaM-binding sites, including an IQ-motif, which has been identified in the C-termini of CNGC20 and CNGC12. Here we present a family-wide analysis of the IQ-motif from all 20 Arabidopsis CNGC isoforms. While most of their IQ-peptides interacted with conserved CaMs in yeast, some were unable to do so, despite high sequence conservation across the family. We showed that the CaM binding ability of the IQ-motif is highly dependent on its proximal and distal vicinity. We determined that two alanine residues positioned N-terminal to the core IQ-sequence play a significant role in CaM binding, and identified a polymorphism at this site that promoted or inhibited CaM binding in yeast. Through detailed biophysical analysis of the CNGC2 IQ-motif, we found that this polymorphism specifically affected the Ca2+-independent interactions with the C-lobe of CaM. This same polymorphism partially suppressed the induction of programmed cell death by CNGC11/12 in planta. Our work expands the model of CNGC regulation, and posits that the C-lobe of apo-CaM is permanently associated with the channel at the N-terminal part of the IQ-domain. This mode allows CaM to function as a Ca2+-sensing regulatory subunit of the channel complex, providing a mechanism by which Ca2+ signals may be fine-tuned., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2017
- Full Text
- View/download PDF
23. Opening the Gates: Insights into Cyclic Nucleotide-Gated Channel-Mediated Signaling.
- Author
-
DeFalco TA, Moeder W, and Yoshioka K
- Subjects
- Calcium metabolism, Calcium Signaling genetics, Calcium Signaling physiology, Cyclic Nucleotide-Gated Cation Channels genetics, Plant Proteins genetics, Cyclic Nucleotide-Gated Cation Channels metabolism, Plant Proteins metabolism
- Abstract
Recent work has expanded our understanding of the roles of cyclic nucleotide-gated channels (CNGCs) in plant signaling. In this spotlight article, we discuss advances and future perspectives in determining how CNGCs mediate calcium signaling in response to diverse stimuli., (Copyright © 2016. Published by Elsevier Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
24. Multiple Calmodulin-Binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12.
- Author
-
DeFalco TA, Marshall CB, Munro K, Kang HG, Moeder W, Ikura M, Snedden WA, and Yoshioka K
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Binding Sites, Calcium metabolism, Calmodulin genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Plant Immunity genetics, Plant Immunity physiology, Protein Binding genetics, Protein Binding physiology, Signal Transduction genetics, Signal Transduction physiology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Calmodulin metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism
- Abstract
Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels., (© 2016 American Society of Plant Biologists. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
25. Crossroads of stress responses, development and flowering regulation--the multiple roles of Cyclic Nucleotide Gated Ion Channel 2.
- Author
-
Fortuna A, Lee J, Ung H, Chin K, Moeder W, and Yoshioka K
- Subjects
- Adaptation, Physiological, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism, Disease Resistance, Flowers metabolism, Gene Expression Regulation, Plant, Mutation, Phenotype, Plant Diseases, Plant Growth Regulators metabolism, Signal Transduction, Arabidopsis genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Flowers growth & development, Salicylic Acid metabolism, Stress, Physiological
- Abstract
The Arabidopsis autoimmune mutant, defense-no death 1 (dnd1) is a null mutant of CYCLIC NUCLEOTIDE-GATED ION CHANNEL2 (AtCNGC2). dnd1 exhibits constitutive pathogen resistance responses including higher levels of endogenous salicylic acid (SA), which is an important signaling molecule for pathogen defense responses. Recently we have reported that dnd1 exhibits a significantly delayed flowering phenotype, indicating the involvement of AtCNGC2 in flowering transition. However, since SA has been known to influence flowering timing as a positive regulator, the delayed flowering phenotype in dnd1 was unexpected. In this study, we have asked whether SA is involved in the dnd1-mediated delayed flowering phenotype. In addition, in order to gain insight into the involvement of SA and CNGCs in flowering transition, we analyzed the flowering transition of cpr22, another CNGC mutant with a similar autoimmune phenotype as dnd1 (including high SA accumulation), and null mutants of several other CNGCs. Our data suggest that dnd1 does not require SA or SA signaling for its delayed flowering phenotype, while SA was responsible for the early flowering phenotype of cpr22. None of the other CNGC mutants besides AtCNGC4 (1) displayed an alteration in flowering transition. This indicates that AtCNGC2 and AtCNGC4 have a unique role controlling flowering timing and this function is independent from its role in pathogen defense.
- Published
- 2015
- Full Text
- View/download PDF
26. Arabidopsis triphosphate tunnel metalloenzyme2 is a negative regulator of the salicylic acid-mediated feedback amplification loop for defense responses.
- Author
-
Ung H, Moeder W, and Yoshioka K
- Subjects
- Arabidopsis microbiology, Down-Regulation, Acid Anhydride Hydrolases metabolism, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Feedback, Pyrophosphatases metabolism, Salicylic Acid metabolism
- Abstract
The triphosphate tunnel metalloenzyme (TTM) superfamily represents a group of enzymes that is characterized by their ability to hydrolyze a range of tripolyphosphate substrates. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes, AtTTM1, AtTTM2, and AtTTM3. Although AtTTM3 has previously been reported to have tripolyphosphatase activity, recombinantly expressed AtTTM2 unexpectedly exhibited pyrophosphatase activity. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. In addition, stronger systemic acquired resistance compared with wild-type plants was observed. These enhanced defense responses are dependent on SA, PHYTOALEXIN-DEFICIENT4, and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. Despite their enhanced pathogen resistance, ttm2 plants did not display constitutively active defense responses, suggesting that AtTTM2 is not a conventional negative regulator but a negative regulator of the amplification of defense responses. The transcriptional suppression of AtTTM2 by pathogen infection or treatment with SA or the systemic acquired resistance activator benzothiadiazole further supports this notion. Such transcriptional regulation is conserved among TTM2 orthologs in the crop plants soybean (Glycine max) and canola (Brassica napus), suggesting that TTM2 is involved in immunity in a wide variety of plant species. This indicates the possible usage of TTM2 knockout mutants for agricultural applications to generate pathogen-resistant crop plants., (© 2014 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2014
- Full Text
- View/download PDF
27. Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development.
- Author
-
Moeder W, Garcia-Petit C, Ung H, Fucile G, Samuel MA, Christendat D, and Yoshioka K
- Subjects
- Acid Anhydride Hydrolases genetics, Arabidopsis genetics, Arabidopsis growth & development, Cell Nucleus genetics, Cell Nucleus metabolism, Crystallography, X-Ray, Meristem enzymology, Meristem genetics, Mutagenesis, Insertional, Mutagenesis, Site-Directed, Plant Roots genetics, Protein Conformation, Acid Anhydride Hydrolases chemistry, Acid Anhydride Hydrolases metabolism, Acid Anhydride Hydrolases physiology, Arabidopsis enzymology, Arabidopsis Proteins chemistry, Arabidopsis Proteins physiology, Plant Roots enzymology, Plant Roots growth & development
- Abstract
The Arabidopsis protein AtTTM3 belongs to the CYTH superfamily named after its two founding members, the CyaB adenylate cyclase from Aeromonas hydrophila and the mammalian thiamine triphosphatase. In this study we report the three-dimensional structure of a plant CYTH domain protein, AtTTM3, determined at 1.9 Å resolution. The crystal structure revealed the characteristic tunnel architecture of CYTH proteins, which specialize in the binding of nucleotides and other organic phosphates and in phosphoryl transfer reactions. The β barrel is composed of eight antiparallel β strands with a cluster of conserved inwardly facing acidic and basic amino acid residues. Mutagenesis of these residues in the catalytic core led to an almost complete loss of enzymatic activity. We established that AtTTM3 is not an adenylate cyclase. Instead, the enzyme displayed weak NTP phosphatase as well as strong tripolyphosphatase activities similar to the triphosphate tunnel metalloenzyme proteins from Clostridium thermocellum (CthTTM) and Nitrosomonas europaea (NeuTTM). AtTTM3 is most highly expressed in the proximal meristematic zone of the plant root. Furthermore, an AtTTM3 T-DNA insertion knockout line displayed a delay in root growth as well as reduced length and number of lateral roots, suggesting a role for AtTTM3 in root development., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)
- Published
- 2013
- Full Text
- View/download PDF
28. The Arabidopsis cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC4 work in the same signaling pathway to regulate pathogen defense and floral transition.
- Author
-
Chin K, DeFalco TA, Moeder W, and Yoshioka K
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins metabolism, Calcium metabolism, Chromosome Segregation genetics, Crosses, Genetic, Models, Biological, Mutation genetics, Peronospora physiology, Phenotype, Plant Diseases immunology, Plant Diseases microbiology, Protein Binding, Protein Subunits metabolism, Suppression, Genetic, Arabidopsis immunology, Arabidopsis microbiology, Cyclic Nucleotide-Gated Cation Channels metabolism, Flowers physiology, Signal Transduction
- Abstract
Arabidopsis (Arabidopsis thaliana) cyclic nucleotide-gated ion channels (CNGCs) form a large family consisting of 20 members and have been implicated in Ca(2+) signaling related to various physiological processes, such as pathogen defense, development, and thermotolerance. The null mutant of AtCNGC2, defense, no death (dnd1), exhibits autoimmune phenotypes, while it is impaired in mounting the hypersensitive response, which is a hallmark of effector-triggered (i.e. RESISTANCE-gene mediated) resistance. It has been suggested that AtCNGC2 is involved in defense responses and likely other aspects of physiology through its role as a Ca(2+)-conducting channel. However, the downstream signaling components and its relation with AtCNGC4, which is the closest paralog of AtCNGC2, remain elusive. Despite the fact that cngc4 mutants display almost identical phenotypes to those seen in cngc2 mutants, not much is known about their relationship. Here, we report the identification and characterization of the Arabidopsis mutant repressor of defense no death1 (rdd1), obtained from a suppressor screen of a transfer DNA insertion knockout mutant of AtCNGC2 in order to identify downstream components of dnd1-mediated signal transduction. rdd1 suppressed the majority of dnd1-mediated phenotypes except Ca(2+) hypersensitivity. In addition, rdd1 also suppressed the dnd1-mediated late-flowering phenotype that was discovered in this study. Our genetic analysis conducted to elucidate the relationship between AtCNGC2 and AtCNGC4 indicates that RDD1 is also involved in AtCNGC4-mediated signal transduction. Lastly, bimolecular fluorescence complementation analysis suggests that AtCNGC2 and AtCNGC4 are likely part of the same channel complex.
- Published
- 2013
- Full Text
- View/download PDF
29. A suppressor screen of the chimeric AtCNGC11/12 reveals residues important for intersubunit interactions of cyclic nucleotide-gated ion channels.
- Author
-
Abdel-Hamid H, Chin K, Moeder W, Shahinas D, Gupta D, and Yoshioka K
- Subjects
- Amino Acid Sequence, Arabidopsis Proteins genetics, Arginine, Base Sequence, Cell Enlargement, Cyclic Nucleotide-Gated Cation Channels genetics, Genes, Suppressor, Genetic Complementation Test, Glycine, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Structure-Activity Relationship, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Cyclic Nucleotide-Gated Cation Channels chemistry, Cyclic Nucleotide-Gated Cation Channels metabolism
- Abstract
To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.
- Published
- 2013
- Full Text
- View/download PDF
30. Forward genetic screening for the improved production of fermentable sugars from plant biomass.
- Author
-
Stamatiou G, Vidaurre DP, Shim I, Tang X, Moeder W, Bonetta D, and McCourt P
- Subjects
- Biological Transport, Carbohydrate Metabolism genetics, Chromosome Mapping, Cluster Analysis, Genes, Plant, Hydrolysis, Indoleacetic Acids metabolism, Mutation, Plants, Genetically Modified, Starch metabolism, Arabidopsis genetics, Arabidopsis metabolism, Biomass, Carbohydrates biosynthesis, Fermentation, Genetic Testing
- Abstract
With their unique metabolism and the potential to produce large amounts of biomass, plants are an excellent bio-energy feedstock for a variety of industrial purposes. Here we developed a high-throughput strategy, using the model plant Arabidopsis thaliana, to identify mutants with improved sugar release from plant biomass. Molecular analysis indicates a variety of processes including starch degradation, cell wall composition and polar transport of the plant hormone auxin can contribute to this improved saccharification. To demonstrate translatability, polar auxin transport in maize was either genetically or chemical inhibited and this also resulted in increased sugar release from plant tissues. Our forward genetic approach using Arabidopsis not only uncovers new functions that contribute to cell wall integrity but also demonstrates that information gleaned from this genetic model can be directly translated to monocotyledonous crops such as maize to improve sugar extractability from biomass.
- Published
- 2013
- Full Text
- View/download PDF
31. High throughput chemical screening supports the involvement of Ca2+ in cyclic nucleotide-gated ion channel-mediated programmed cell death in Arabidopsis.
- Author
-
Abdel-Hamid H, Chin K, Moeder W, and Yoshioka K
- Subjects
- Arabidopsis genetics, Calcium metabolism, Gene Knockout Techniques, Gravitropism, High-Throughput Screening Assays, Mutation, Plant Immunity, Potassium metabolism, Apoptosis, Arabidopsis physiology, Arabidopsis Proteins physiology, Calcium Signaling, Cyclic Nucleotide-Gated Cation Channels physiology
- Abstract
Recently, we reported the role of Arabidopsis cyclic nucleotide-gated ion channel (AtCNGC) 11 and 12 in Ca2+-dependent physiological responses. AtCNGC11 and 12 have been reported to be involved in plant immunity, but whether these channels play additional physiological roles was not clear before. Using single and double knockout mutants, we have found that these channels play significant roles in Ca2+ signaling, which mediates several physiological processes, such as gravitropic bending and senescence. Here, we conducted a high throughput, non-biased chemical screen using the gain-of-function mutant of AtCNGC11 and 12, cpr22. Our data presented here indicates that Ca2+ but not K+ channel blockers suppress AtCNGC11/12-induced lethality. Our data further suggest that AtCNGC11 and 12 are involved in Ca2+-dependent, but not K+-dependent physiological responses in planta.
- Published
- 2011
- Full Text
- View/download PDF
32. The cyclic nucleotide-gated channels AtCNGC11 and 12 are involved in multiple Ca²⁺-dependent physiological responses and act in a synergistic manner.
- Author
-
Urquhart W, Chin K, Ung H, Moeder W, and Yoshioka K
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Cellular Senescence genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Gravitropism genetics, Gravitropism physiology, Plants, Genetically Modified genetics, Promoter Regions, Genetic genetics, Tandem Repeat Sequences genetics, Arabidopsis metabolism, Arabidopsis physiology, Arabidopsis Proteins metabolism, Calcium metabolism, Cellular Senescence physiology, Cyclic Nucleotide-Gated Cation Channels metabolism, Plants, Genetically Modified metabolism
- Abstract
Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. These channels have so far been reported to be involved in a diverse range of physiological phenomena. For example, AtCNGC18 was reported to play an important role in pollen tube growth, while AtCNGC2, 4, 11, and 12 were implicated in mediating pathogen defence. To identify additional functions for AtCNGC11 and 12, various physiological aspects were analysed using both AtCNGC11 and 12 single knockout mutants as well as a double mutant. Although AtCNGC11 and 12 can function as K(+) and Ca(2+) channels in yeast, it was found that the loss of AtCNGC11 and 12 in Arabidopsis caused increased sensitivity to Ca(2+) but not K(+), indicating a specific function for these genes in Ca(2+) signalling in planta. However, they did not show an alteration in Ca(2+) accumulation, suggesting that AtCNGC11 and 12 are not involved in general Ca(2+) homeostasis but rather in the endogenous movement of Ca(2+) and/or Ca(2+) signalling. Furthermore, these channels synergistically contribute to the generation of a Ca(2+) signal that leads to gravitropic bending. Finally, AtCNGC11 and 12 gene expression was induced during dark-induced senescence and AtCNGC11 and 12 knockout mutants displayed enhanced chlorophyll loss, which was even more pronounced in the double mutant, also indicating synergistic roles in senescence. The findings indicate that (i) some CNGC family members have multiple physiological functions and (ii) some plant CNGCs share the same biological function and work in a synergistic manner.
- Published
- 2011
- Full Text
- View/download PDF
33. The role of cyclic nucleotide-gated ion channels in plant immunity.
- Author
-
Moeder W, Urquhart W, Ung H, and Yoshioka K
- Subjects
- Cyclic Nucleotide-Gated Cation Channels genetics, Phenotype, Plant Diseases genetics, Plant Diseases immunology, Plant Diseases microbiology, Plant Immunity genetics, Plants genetics, Plants immunology, Plants microbiology, Signal Transduction immunology, Cyclic Nucleotide-Gated Cation Channels metabolism, Plant Immunity immunology
- Abstract
Since the first plant cyclic nucleotide-gated ion channel (CNGC), HvCBT1, was identified as a calmodulin binding protein, more than a decade has passed and a substantial amount of work has been done to understand the molecular nature and function of these channel proteins. Based on electrophysiological and heterologous expression analyses, plant CNGCs function as non-selective cation channels and, so far, their biological roles have been reported in defense responses, development, and ion homeostasis. Forward genetic approaches identified four AtCNGCs (AtCNGC2, 4, 11, and 12) to be involved in plant immunity, as null mutants for AtCNGC2, 4, 11, and 12 as well as a gain-of- function mutant for AtCNGC11 and 12 exhibited alterations in defense responses. Since ion flux changes have been reported as one of the early events upon pathogen recognition and also are an essential component for the activation of defense responses, the involvement of CNGCs in these ion flux changes has been suggested. However, the recent detailed characterization of null mutants suggested a more complex involvement of this channel family. In this review, we focus on the discoveries and characterization of these CNGC mutants and discuss possible roles of CNGCs as components in plant immunity.
- Published
- 2011
- Full Text
- View/download PDF
34. Altered germination and subcellular localization patterns for PUB44/SAUL1 in response to stress and phytohormone treatments.
- Author
-
Salt JN, Yoshioka K, Moeder W, and Goring DR
- Subjects
- Arabidopsis drug effects, Arabidopsis genetics, Arabidopsis Proteins genetics, Gene Expression Regulation, Plant drug effects, Gene Expression Regulation, Plant genetics, Germination genetics, Plants, Genetically Modified drug effects, Plants, Genetically Modified genetics, Ubiquitin-Protein Ligases genetics, Abscisic Acid pharmacology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Germination drug effects, Plant Growth Regulators pharmacology, Plants, Genetically Modified metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Background: In plants, the ubiquitin-proteasome system is emerging as a significant regulatory system throughout the plant lifecycle. The ubiquitination of a target protein requires the sequential actions of the E1, E2 and E3 enzymes, with the latter E3 enzyme conferring target selection in this process. There are a large number of predicted E3 enzymes in plant genomes, and very little is known about the functions of many of these predicted genes. Here we report here an analysis of two closely-related members of the Arabidopsis Plant U-box (PUB) family of E3 ubiquitin ligases, PUB43 and PUB44., Principal Findings: Homozygous pub44/pub44 mutant seedlings were found displayed a seedling lethal phenotype and this corresponded with widespread cell death lesions throughout the cotyledons and roots. Interestingly, heterozygous PUB44/pub44 seedlings were wild-type in appearance yet displayed intermediate levels of cell death lesions in comparison to pub44/pub44 seedlings. In contrast, homozygous pub43/pub43 mutants were viable and did not show any signs of cell death despite the PUB43 gene being more highly expressed than PUB44. The PUB44 mutants are not classical lesion mimic mutants as they did not have increased resistance to plant pathogens. We also observed increased germination rates in mutant seeds for both PUB44 and PUB43 under inhibitory concentrations of abscisic acid. Finally, the subcellular localization of PUB44 was investigated with transient expression assays in BY-2 cells. Under varying conditions, PUB44 was observed to be localized to the cytoplasm, plasma membrane, or nucleus., Conclusions: Based on mutant plant analyses, the Arabidopsis PUB43 and PUB44 genes are proposed to function during seed germination and early seedling growth. Given PUB44's ability to shuttle from the nucleus to the plasma membrane, PUB44 may be active in different subcellular compartments as part of these biological functions.
- Published
- 2011
- Full Text
- View/download PDF
35. Leaf senescence signaling: the Ca2+-conducting Arabidopsis cyclic nucleotide gated channel2 acts through nitric oxide to repress senescence programming.
- Author
-
Ma W, Smigel A, Walker RK, Moeder W, Yoshioka K, and Berkowitz GA
- Subjects
- Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Cyclic Nucleotide-Gated Cation Channels genetics, Darkness, Gene Expression Regulation, Plant, Hydrogen Peroxide analysis, Lipid Peroxidation, Mutation, Arabidopsis genetics, Calcium metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism, Nitric Oxide metabolism, Plant Leaves physiology
- Abstract
Ca(2+) and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca(2+). The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca(2+) modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca(2+) and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca(2+) uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca(2+) accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H(2)O(2) generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca(2+) channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca(2+) signaling provides evidence consistent with genetic studies of the relationship between Ca(2+) signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca(2+) uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.
- Published
- 2010
- Full Text
- View/download PDF
36. SA-ABA antagonism in defense responses.
- Author
-
Moeder W, Ung H, Mosher S, and Yoshioka K
- Subjects
- Abscisic Acid metabolism, Arabidopsis genetics, Desiccation, Genes, Plant genetics, Humidity, Immunity, Innate immunology, Phenotype, Plant Diseases immunology, Salicylic Acid metabolism, Signal Transduction, Abscisic Acid antagonists & inhibitors, Arabidopsis immunology, Arabidopsis metabolism, Salicylic Acid antagonists & inhibitors
- Abstract
Until recently, phytohormones were mostly studied separately. However, recent studies have suggested that the signaling pathways involved are highly interconnected. We recently reported the antagonistic effects of salicylic acid (SA) and abscisic acid (ABA) in the lesion mimic mutants, cpr22 and ssi4. After shifting these mutants from high humidity, where the lesion mimic phenotype is suppressed to permissive low humidity condition, both SA and ABA pathways were up-regulated. However, the increased levels of SA were able to block downstream ABA responses even though ABA signaling genes and endogenous ABA were elevated. Furthermore, these lesion mimic mutants displayed a partial ABA insensitivity with respect to germination, guard cell opening, and water loss. This increased water loss in detached mutant plants could also be mimicked by treating wild type plants with SA. An active SA analog, 5-chloro-salicylic acid also induced enhanced water loss, while an inactive analog, 4-hydroxy-benzoic acid, did not. Here, we report that the biological analogs of SA, the systemic acquired resistance (SAR) activators, BTH (benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester) and BIT (1,2-benzisothiazol-3(2H)-one1,1-dioxide), did not have the same effect as SA, suggesting that SA may have additional roles to defense, and that SAR activators may not mimic all SA effects., (© 2010 Landes Bioscience)
- Published
- 2010
- Full Text
- View/download PDF
37. Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels.
- Author
-
Abdel-Hamid H, Chin K, Shahinas D, Moeder W, and Yoshioka K
- Subjects
- Amino Acid Sequence, Arabidopsis chemistry, Arabidopsis genetics, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Binding Sites, Calmodulin chemistry, Cyclic Nucleotide-Gated Cation Channels chemistry, Cyclic Nucleotide-Gated Cation Channels genetics, Mutation, Protein Binding, Protein Conformation, Sequence Alignment, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Calmodulin metabolism, Cyclic Nucleotide-Gated Cation Channels metabolism
- Abstract
Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.
- Published
- 2010
- Full Text
- View/download PDF
38. Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis.
- Author
-
Chin K, Moeder W, Abdel-Hamid H, Shahinas D, Gupta D, and Yoshioka K
- Subjects
- Amino Acid Sequence, Arabidopsis chemistry, Arabidopsis genetics, Arabidopsis Proteins genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Molecular Conformation, Molecular Sequence Data, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Cyclic Nucleotide-Gated Cation Channels chemistry, Cyclic Nucleotide-Gated Cation Channels metabolism, Gene Expression Regulation, Plant
- Abstract
The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.
- Published
- 2010
- Full Text
- View/download PDF
39. The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner.
- Author
-
Mosher S, Moeder W, Nishimura N, Jikumaru Y, Joo SH, Urquhart W, Klessig DF, Kim SK, Nambara E, and Yoshioka K
- Subjects
- Genes, Plant, Abscisic Acid metabolism, Arabidopsis microbiology, Molecular Mimicry, Salicylic Acid pharmacology, Signal Transduction
- Abstract
A number of Arabidopsis (Arabidopsis thaliana) lesion-mimic mutants exhibit alterations in both abiotic stress responses and pathogen resistance. One of these mutants, constitutive expresser of PR genes22 (cpr22), which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion-mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent. These defense responses in cpr22 are suppressed under high-humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not the wild type, showed a rapid increase in SA levels followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were up-regulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, was down-regulated, but that of other genes, like ABI1 and HAB1, was up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA not only in abiotic stress responses but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together, these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.
- Published
- 2010
- Full Text
- View/download PDF
40. Forward and reverse genetics to identify genes involved in the age-related resistance response in Arabidopsis thaliana.
- Author
-
Carviel JL, Al-Daoud F, Neumann M, Mohammad A, Provart NJ, Moeder W, Yoshioka K, and Cameron RK
- Subjects
- Arabidopsis immunology, Arabidopsis microbiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Gene Expression Profiling, Gene Expression Regulation, Plant drug effects, Immunity, Innate immunology, Intracellular Space drug effects, Intracellular Space metabolism, Mutation genetics, Oligonucleotide Array Sequence Analysis, Peronospora drug effects, Peronospora growth & development, Plant Diseases microbiology, Pseudomonas syringae drug effects, Salicylic Acid metabolism, Salicylic Acid pharmacology, Arabidopsis genetics, Arabidopsis growth & development, Genes, Plant, Genetic Techniques, Immunity, Innate genetics, Plant Diseases genetics, Plant Diseases immunology
- Abstract
SUMMARY Age-related resistance (ARR) occurs in numerous plant species, often resulting in increased disease resistance as plants mature. ARR in Arabidopsis to Pseudomonas syringae pv. tomato is associated with intercellular salicylic acid (SA) accumulation and the transition to flowering. Forward and reverse genetic screens were performed to identify genes required for ARR and to investigate the mechanism of the ARR response. Infiltration of SA into the intercellular space of the ARR-defective mutant iap1-1 (important for the ARR pathway) partially restored ARR function. Inter- and intracellular SA accumulation was reduced in the mutant iap1-1 compared with the wild-type, and the SA regulatory gene EDS1 was also required for ARR. Combining microarray analysis with reverse genetics using T-DNA insertion lines, four additional ARR genes were identified as contributing to ARR: two plant-specific transcription factors of the NAC family [ANAC055 (At3g15500) and ANAC092 (At5g39610)], a UDP-glucose glucosyltransferase [UGT85A1 (At1g22400)] and a cytidine deaminase [CDA1 (At2g19570)]. These four genes and IAP1 are also required for ARR to Hyaloperonospora parasitica. IAP1 encodes a key component of ARR that acts upstream of SA accumulation and possibly downstream of UGT85A1, CDA1 and the two NAC transcription factors (ANAC055, ANAC092).
- Published
- 2009
- Full Text
- View/download PDF
41. Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene.
- Author
-
Baxter J, Moeder W, Urquhart W, Shahinas D, Chin K, Christendat D, Kang HG, Angelova M, Kato N, and Yoshioka K
- Subjects
- Amino Acid Sequence, Amino Acids genetics, Arabidopsis chemistry, Arabidopsis Proteins genetics, Crosses, Genetic, Cyclic Nucleotide-Gated Cation Channels genetics, Genes, Plant, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Mutant Chimeric Proteins chemistry, Mutant Chimeric Proteins genetics, Mutation, Phenotype, Plants, Genetically Modified chemistry, Plants, Genetically Modified genetics, Plasmids, Protein Structure, Secondary, RNA, Plant genetics, Reverse Transcriptase Polymerase Chain Reaction, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Sequence Alignment, Structure-Activity Relationship, Nicotiana chemistry, Nicotiana genetics, Amino Acids chemistry, Arabidopsis genetics, Arabidopsis Proteins chemistry, Cyclic Nucleotide-Gated Cation Channels chemistry
- Abstract
We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.
- Published
- 2008
- Full Text
- View/download PDF
42. Lesion mimic mutants: A classical, yet still fundamental approach to study programmed cell death.
- Author
-
Moeder W and Yoshioka K
- Abstract
Over the last decade a substantial number of lesion mimic mutants (LMM) have been isolated and a growing number of the genes have been cloned. It is now becoming clear that these mutants are valuable tools to dissect various aspects of programmed cell death (PCD) and pathogen resistance pathways in plants. Together with other forward genetics approaches LMMs shed light on the PCD machinery in plant cells and revealed important roles for sphingolipids, Ca(2+) and chloroplast-derived porphyrin-metabolites during cell death development.
- Published
- 2008
- Full Text
- View/download PDF
43. The chimeric cyclic nucleotide-gated ion channel ATCNGC11/12 constitutively induces programmed cell death in a Ca2+ dependent manner.
- Author
-
Urquhart W, Gunawardena AH, Moeder W, Ali R, Berkowitz GA, and Yoshioka K
- Subjects
- Apoptosis drug effects, Arabidopsis cytology, Arabidopsis metabolism, Arabidopsis Proteins analysis, Arabidopsis Proteins physiology, Calcium Channels analysis, Calcium Channels physiology, Caspase Inhibitors, Caspases physiology, Cyclic Nucleotide-Gated Cation Channels analysis, Cyclic Nucleotide-Gated Cation Channels physiology, Cysteine Endopeptidases physiology, Cysteine Proteinase Inhibitors pharmacology, DNA Fragmentation, Mutant Chimeric Proteins analysis, Plants, Genetically Modified cytology, Plants, Genetically Modified drug effects, Plants, Genetically Modified metabolism, Nicotiana genetics, Apoptosis physiology, Arabidopsis Proteins genetics, Calcium Channels genetics, Calcium Signaling, Cyclic Nucleotide-Gated Cation Channels genetics, Mutant Chimeric Proteins physiology
- Abstract
The hypersensitive response (HR) involves programmed cell death (PCD) in response to pathogen infection. To investigate the pathogen resistance signaling pathway, we previously identified the Arabidopsis mutant cpr22, which displays constitutive activation of multiple defense responses including HR like cell death. The cpr22 mutation has been identified as a 3 kb deletion that fuses two cyclic nucleotide-gated ion channel (CNGC)-encoding genes, ATCNGC11 and ATCNGC12, to generate a novel chimeric gene, ATCNGC11/12. In this study, we conducted a characterization of cell death induced by transient expression of ATCNGC11/12 in Nicotiana benthamiana. Electron microscopic analysis of this cell death showed similar characteristics to PCD, such as plasma membrane shrinkage and vesicle formation. The hallmark of animal PCD, fragmentation of nuclear DNA, was also observed in ATCNGC11/12-induced cell death. The development of cell death was significantly suppressed by caspase-1 inhibitors, suggesting the involvement of caspases in this process. Recently, vacuolar processing enzyme (VPE) was isolated as the first plant caspase-like protein, which is involved in HR development. In VPE-silenced plants development of cell death induced by ATCNGC11/12 was much slower and weaker compared to control plants, suggesting the involvement of VPE as a caspase in ATCNGC11/12-induced cell death. Complementation analysis using a Ca2+ uptake deficient yeast mutant demonstrated that the ATCNGC11/12 channel is permeable to Ca2+. Additionally, calcium channel blockers such as GdCl3 inhibited ATCNGC11/12-induced HR formation, whereas potassium channel blockers did not. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that the chimeric channel, ATCNGC11/12, is at the point of, or up-stream of the calcium signal necessary for the development of HR.
- Published
- 2007
- Full Text
- View/download PDF
44. Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana.
- Author
-
Moeder W, Del Pozo O, Navarre DA, Martin GB, and Klessig DF
- Subjects
- Arabidopsis metabolism, Nicotiana metabolism, Aconitate Hydratase physiology, Arabidopsis cytology, Cell Death physiology, Oxidative Stress physiology, Nicotiana cytology
- Abstract
In animals, aconitase is a bifunctional protein. When an iron-sulfur cluster is present in its catalytic center, aconitase displays enzymatic activity; when this cluster is lost, it switches to an RNA-binding protein that regulates the translatability or stability of certain transcripts. To investigate the role of aconitase in plants, we assessed its ability to bind mRNA. Recombinant aconitase failed to bind an iron responsive element (IRE) from the human ferritin gene. However, it bound the 5' UTR of the Arabidopsis chloroplastic CuZn superoxide dismutase 2 (CSD2) mRNA, and this binding was specific. Arabidopsis aconitase knockout (KO) plants were found to have significantly less chlorosis after treatment with the superoxide-generating compound, paraquat. This phenotype correlated with delayed induction of the antioxidant gene GST1, suggesting that these KO lines are more tolerant to oxidative stress. Increased levels of CSD2 mRNAs were observed in the KO lines, although the level of CSD2 protein was not affected. Virus-induced gene silencing (VIGS) of aconitase in Nicotiana benthamiana caused a 90% reduction in aconitase activity, stunting, spontaneous necrotic lesions, and increased resistance to paraquat. The silenced plants also had less cell death after transient co-expression of the AvrPto and Pto proteins or the pro-apoptotic protein Bax. Following inoculation with Pseudomonas syringae pv. tabaci carrying avrPto, aconitase-silenced N. benthamiana plants expressing the Pto transgene displayed a delayed hypersensitive response (HR) and supported higher levels of bacterial growth. Disease-associated cell death in N. benthamiana inoculated with P. s. pv. tabaci was also reduced. Taken together, these results suggest that aconitase plays a role in mediating oxidative stress and regulating cell death.
- Published
- 2007
- Full Text
- View/download PDF
45. NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes.
- Author
-
Oide S, Moeder W, Krasnoff S, Gibson D, Haas H, Yoshioka K, and Turgeon BG
- Subjects
- Ascomycota pathogenicity, Base Sequence, Conserved Sequence, DNA Primers, Molecular Sequence Data, Oxidative Stress, Peptide Synthases chemistry, Siderophores biosynthesis, Virulence, Ascomycota enzymology, Iron metabolism, Peptide Synthases metabolism, Plants microbiology, Siderophores metabolism
- Abstract
NPS6, encoding a nonribosomal peptide synthetase, is a virulence determinant in the maize (Zea mays) pathogen Cochliobolus heterostrophus and is involved in tolerance to H(2)O(2). Deletion of NPS6 orthologs in the rice (Oryza sativa) pathogen, Cochliobolus miyabeanus, the wheat (Triticum aestivum) pathogen, Fusarium graminearum, and the Arabidopsis thaliana pathogen, Alternaria brassicicola, resulted in reduced virulence and hypersensitivity to H(2)O(2). Introduction of the NPS6 ortholog from the saprobe Neurospora crassa to the Deltanps6 strain of C. heterostrophus restored wild-type virulence to maize and tolerance to H(2)O(2), demonstrating functional conservation in filamentous ascomycete phytopathogens and saprobes. Increased sensitivity to iron depletion was identified as a conserved phenotype of Deltanps6 strains. Exogenous application of iron enhanced the virulence of Deltanps6 strains of C. heterostrophus, C. miyabeanus, F. graminearum, and A. brassicicola to each host. NPS6 is responsible for the biosynthesis of extracellular siderophores by C. heterostrophus, F. graminearum, and A. brassicicola. Application of the extracellular siderophore of A. brassicicola restored wild-type virulence of the DeltaAbnps6 strain to Arabidopsis. It is proposed that the role of extracellular siderophores in fungal virulence to plants is to supply an essential nutrient, iron, to their producers in planta and not to act as phytotoxins, depriving their hosts of iron.
- Published
- 2006
- Full Text
- View/download PDF
46. The chimeric Arabidopsis CYCLIC NUCLEOTIDE-GATED ION CHANNEL11/12 activates multiple pathogen resistance responses.
- Author
-
Yoshioka K, Moeder W, Kang HG, Kachroo P, Masmoudi K, Berkowitz G, and Klessig DF
- Subjects
- Amino Acid Sequence, Arabidopsis anatomy & histology, Arabidopsis Proteins metabolism, Arabidopsis Proteins physiology, Base Sequence, Chromosomes, Plant genetics, Cloning, Molecular, Cyclic Nucleotide-Gated Cation Channels, Immunity, Innate physiology, Ion Channels physiology, Molecular Sequence Data, Mutant Chimeric Proteins genetics, Mutant Chimeric Proteins metabolism, Mutation, Phenotype, Physical Chromosome Mapping, Plant Diseases microbiology, Plants, Genetically Modified genetics, Plants, Genetically Modified metabolism, Plants, Genetically Modified microbiology, Sequence Alignment, Sequence Deletion, Signal Transduction, Nicotiana genetics, Arabidopsis genetics, Arabidopsis microbiology, Arabidopsis Proteins genetics, Ion Channels genetics, Mutant Chimeric Proteins physiology
- Abstract
To investigate the resistance signaling pathways activated by pathogen infection, we previously identified the Arabidopsis thaliana mutant constitutive expresser of PR genes22 (cpr22), which displays constitutive activation of multiple defense responses. Here, we identify the cpr22 mutation as a 3-kb deletion that fuses two cyclic nucleotide-gated ion channel (ATCNGC)-encoding genes, ATCNGC11 and ATCNGC12, to generate a novel chimeric gene, ATCNGC11/12. Genetic, molecular, and complementation analyses suggest that ATCNGC11/12, as well as ATCNGC11 and ATCNGC12, form functional cAMP-activated ATCNGCs and that the phenotype conferred by cpr22 is attributable to the expression of ATCNGC11/12. However, because overexpression of ATCNGC12, but not ATCNGC11, suppressed the phenotype conferred by cpr22, the development of this phenotype appears to be regulated by the ratio between ATCNGC11/12 and ATCNGC12. Analysis of knockout lines revealed that both ATCNGC11 and ATCNGC12 are positive mediators of resistance against an avirulent biotype of Hyaloperonospora parasitica. Through epistatic analyses, cpr22-mediated enhanced resistance to pathogens was found to require NDR1-dependent and EDS1/PAD4-dependent pathways. In striking contrast, none of these pathways was required for cpr22-induced salicylic acid accumulation or PR-1 gene expression. These results demonstrate that NDR1, EDS1, and PAD4 mediate other resistance signaling function(s) in addition to salicylic acid and pathogenesis-related protein accumulation. Moreover, the requirement for both NDR1-dependent and EDS1/PAD4-dependent pathways for cpr22-mediated resistance suggests that these pathways are cross-regulated.
- Published
- 2006
- Full Text
- View/download PDF
47. Arabidopsis ssi2-conferred susceptibility to Botrytis cinerea is dependent on EDS5 and PAD4.
- Author
-
Nandi A, Moeder W, Kachroo P, Klessig DF, and Shah J
- Subjects
- Arabidopsis microbiology, Cell Death, Cyclopentanes metabolism, Gene Expression Regulation, Plant, Oxylipins, Plant Diseases genetics, Plant Diseases microbiology, Plant Leaves genetics, Plant Leaves metabolism, Pseudomonas syringae pathogenicity, Salicylic Acid metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, Botrytis pathogenicity, Carboxylic Ester Hydrolases genetics, Fatty Acid Desaturases genetics, Membrane Transport Proteins genetics
- Abstract
Loss of a stearoyl-ACP desaturase activity in the Arabidopsis thaliana ssi2 mutant confers susceptibility to the necrotroph, Botrytis cinerea. In contrast, the ssi2 mutant exhibits enhanced resistance to Pseudomonas syringae, Peronospora parasitica, and Cucumber mosaic virus. The altered basal resistance to these pathogens in the ssi2 mutant plant is accompanied by the constitutive accumulation of elevated salicylic acid (SA) level and expression of the pathogenesis-related 1 (PR1) gene, the inability of jasmonic acid (JA) to activate expression of the defensin gene, PDF1.2, and the spontaneous death of cells. Here, we show that presence of the eds5 and pad4 mutant alleles compromises the ssi2-conferred resistance to Pseudomonas syringae pv. maculicola. In contrast, resistance to B. cinerea was restored in the ssi2 eds5 and ssi2 pad4 double-mutant plants. However, resistance to B. cinerea was not accompanied by the restoration of JA responsiveness in the ssi2 eds5 and ssi2 pad4 plants. The ssi2 eds5 and ssi2 pad4 plants retain the ssi2-conferred spontaneous cell death phenotype, suggesting that cell death is not a major factor that predisposes the ssi2 mutant to infection by B. cinerea. Furthermore, the high SA content of the ssi2 pad4 plant, combined with our previous observation that the SA-deficient ssi2 nahG plant succumbs to infection by B. cinerea, suggests that elevated SA level does not have a causal role in the ssi2-conferred susceptibility to B. cinerea. Our results suggest that interaction between an SSI2-dependent factor or factors and an EDS5- and PAD4-dependent mechanism or mechanisms modulates defense to B. cinerea.
- Published
- 2005
- Full Text
- View/download PDF
48. Involvement of the small GTPase Rac in the defense responses of tobacco to pathogens.
- Author
-
Moeder W, Yoshioka K, and Klessig DF
- Subjects
- Gene Expression Regulation, Plant, Immunity, Innate, Plant Diseases microbiology, Plant Diseases virology, Plants, Genetically Modified, Pseudomonas syringae, Reactive Oxygen Species, Respiratory Burst, Salicylic Acid metabolism, Nicotiana enzymology, Nicotiana microbiology, Nicotiana virology, Tobacco Mosaic Virus, rac1 GTP-Binding Protein genetics, Plant Proteins physiology, Nicotiana physiology, rac GTP-Binding Proteins physiology
- Abstract
During the hypersensitive response (HR), plants accumulate reactive oxygen species (ROS) that are likely generated at least in part by an NADPH oxidase similar to that found in mammalian neutrophils. An essential regulator of mammalian NADPH oxidase is the small GTP-binding protein Rac. To investigate whether Rac also regulates the pathogen-induced oxidative burst in plants, a dominant negative form of the rice OsRac1 gene was overexpressed in tobacco carrying the N resistance gene. Following infection with Tobacco mosaic virus (TMV), DN-OsRacl plants developed smaller lesions than wild-type plants, accumulated lower levels of lipid peroxidation products, and failed to activate expression of antioxidant genes. These results, combined with the demonstration that superoxide and hydrogen peroxide levels were reduced in DN-OsRacl tobacco developing a synchronous HR triggered by transient expression of the TMV p50 helicase domain or the Pto and AvrPto proteins, suggest that ROS production is impaired. The dominant negative effect of DN-OsRacl could be rescued by transiently overexpressing the wild-type OsRac1 protein. TMV-induced salicylic acid accumulation also was compromised in DN-OsRacl tobacco. Interestingly, while systemic acquired resistance to TMV was not impaired, nonhost resistance to Pseudomonas syringae pv. maculicola ES4326 was suppressed. Thus, the effect DN-OsRac1 expression exerts on the resistance signaling pathway appears to vary depending on the identity of the inoculated pathogen.
- Published
- 2005
- Full Text
- View/download PDF
49. The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1.
- Author
-
Barth C, Moeder W, Klessig DF, and Conklin PL
- Subjects
- Arabidopsis genetics, Arabidopsis microbiology, Gene Expression Regulation, Plant, Immunity, Innate, Mutation, Plant Diseases genetics, Plant Diseases microbiology, Time Factors, Transcription, Genetic genetics, Arabidopsis physiology, Ascorbic Acid metabolism, Ascorbic Acid Deficiency metabolism, Pseudomonas growth & development
- Abstract
The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses.
- Published
- 2004
- Full Text
- View/download PDF
50. The Pseudomonas syringae type III effector AvrRpt2 functions downstream or independently of SA to promote virulence on Arabidopsis thaliana.
- Author
-
Chen Z, Kloek AP, Cuzick A, Moeder W, Tang D, Innes RW, Klessig DF, McDowell JM, and Kunkel BN
- Subjects
- Arabidopsis metabolism, Arabidopsis microbiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Bacterial Proteins metabolism, Fungi growth & development, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Plant, Immunity, Innate genetics, Mixed Function Oxygenases genetics, Mixed Function Oxygenases metabolism, Mutation, Peronospora growth & development, Plant Diseases microbiology, Plant Proteins genetics, Plant Proteins metabolism, Plants, Genetically Modified, Signal Transduction genetics, Virulence genetics, Arabidopsis genetics, Bacterial Proteins genetics, Pseudomonas syringae pathogenicity, Salicylic Acid metabolism
- Abstract
AvrRpt2, a Pseudomonas syringae type III effector protein, functions from inside plant cells to promote the virulence of P. syringae pv. tomato strain DC3000 (PstDC3000) on Arabidopsis thaliana plants lacking a functional copy of the corresponding RPS2 resistance gene. In this study, we extended our understanding of AvrRpt2 virulence activity by exploring the hypothesis that AvrRpt2 promotes PstDC3000 virulence by suppressing plant defenses. When delivered by PstDC3000, AvrRpt2 suppresses pathogen-related (PR) gene expression during infection, suggesting that AvrRpt2 suppresses defenses mediated by salicylic acid (SA). However, AvrRpt2 promotes PstDC3000 growth on transgenic plants expressing the SA-degrading enzyme NahG, indicating that AvrRpt2 does not promote bacterial virulence by modulating SA levels during infection. AvrRpt2 general virulence activity does not depend on the RPM1 resistance gene, as mutations in RPM1 had no effect on AvrRpt2-induced phenotypes. Transgenic plants expressing AvrRpt2 displayed enhanced susceptibility to PstDC3000 strains defective in type III secretion, indicating that enhanced susceptibility of these plants is not because of suppression of defense responses elicited by other type III effectors. Additionally, avrRpt2 transgenic plants did not exhibit increased susceptibility to Peronospora parasitica and Erysiphe cichoracearum, suggesting that AvrRpt2 virulence activity is specific to P. syringae.
- Published
- 2004
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.