scholarly journals Decreased Inositol 1,4,5-Trisphosphate Content in Pathogen-Challenged Soybean Cells

2000 ◽  
Vol 13 (5) ◽  
pp. 563-567 ◽  
Author(s):  
Toshiro Shigaki ◽  
Madan K. Bhattacharyya
Keyword(s):  
Phospholipase C ◽  
Pseudomonas Syringae ◽  
Bacterial Pathogen ◽  
Plant Cells ◽  
Water Control ◽  
Inositol 1,4,5 Trisphosphate ◽  
Infected Cells ◽  
Related Compounds ◽  
Incompatible Interactions

Phosphoinositide-specific phospholipase C (PI-PLC) has been shown to be transiently activated when plant cells were treated with elicitors. We thus investigated the activity of PI-PLC when soybean cells were infected with the bacterial pathogen Pseudomonas syringae pv. glycinea, by measuring cellular cytosolic inositol 1,4,5-trisphosphate (IP3) levels. We observed that IP3 content decreased in both compatible and incompatible interactions. In vitro phosphatase activities were similar in both water control and infected cells with slightly lower IP3 degradation observed for infected cells, indicating that the reduced IP3 content in infected cells most likely results from reduced PI-PLC activity. We hypothesize that reduced IP3 content following infection may lead to suppression of various housekeeping activities of the cells, thus diverting the cellular resources either to the synthesis of defense-related compounds against pathogens, and/or to the growth of pathogens.

scholarly journals Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical for Listeria monocytogenes Virulence

2006 ◽  
Vol 75 (1) ◽  
pp. 44-51 ◽  
Author(s):  
P. S. Marie Yeung ◽  
Yoojin Na ◽  
Amanda J. Kreuder ◽  
Hélène Marquis
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Membrane Damage ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Virulence Attenuation ◽  
Infected Cells ◽  
Host Cell Membranes ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBΔpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBΔpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

scholarly journals Molecular characterization of differences between the tomato immune receptors Fls3 and Fls2

2020 ◽  
Author(s):  
Robyn Roberts ◽  
Alexander E. Liu ◽  
Lingwei Wan ◽  
Annie M. Geiger ◽  
Sarah R. Hind ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Molecular Mechanisms ◽  
Bacterial Pathogen ◽  
Transcript Abundance ◽  
Defense Responses ◽  
Structural Features ◽  
Host Responses ◽  
Pathogen Invasion

AbstractPlants mount defense responses by recognizing indications of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) contains two MAMPs, flg22 and flgII-28, that are recognized by tomato receptors Flagellin sensing 2 (Fls2) and Flagellin sensing 3 (Fls3), respectively. It is unknown to what degree each receptor contributes to immunity and if they promote immune responses using the same molecular mechanisms. Characterization of CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants revealed that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, striking differences were observed in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species (ROS) and an increase in transcript abundance of 44 tomato genes, with two genes serving as reporters for Fls3. Fls3 had greater in vitro kinase activity and interacted differently with the Pst effector AvrPtoB as compared to Fls2. Using chimeric Fls2/Fls3 proteins, we found that no receptor domain was solely responsible for the Fls3 sustained ROS, suggesting involvement of multiple structural features. This work reveals differences in the immunity outputs between Fls2 and Fls3, suggesting they use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

scholarly journals The ecological genetics ofPseudomonas syringaefrom kiwifruit leaves

2017 ◽  
Author(s):  
Christina Straub ◽  
Elena Colombi ◽  
Li Li ◽  
Hongwen Huang ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Population Structure ◽  
Pseudomonas Syringae ◽  
Bacterial Pathogen ◽  
Ecological Factors ◽  
Ecological Genetics ◽  
Plant Interactions ◽  
In Planta ◽  
Canker Disease ◽  
Pathogen Populations

SUMMARYInteractions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurringPseudomonas syringaeisolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused byP. syringaepv.actinidiae(Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenicPsaand commensalP. syringaePG3a isolated from the same leaf was tested using reciprocal invasion from rare assaysin vitroand in planta.P. syringaeG33C (PG3a) inhibitedPsaNZ54, while the presence ofPsaNZ54 enhanced the growth ofP. syringaeG33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system ofPsa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations.ORIGINALITY-SIGNIFICANT STATEMENTBacterial pathogen populations are often studied with little consideration of co-occurring microbes and yet interactions between pathogens and commensals can affect both population structure and disease progression. A fine-scale sampling of commensals present on kiwifruit leaves during an outbreak of bleeding canker disease caused byP. syringaepv.actinidiaereveals a clonal population structure. A new clade of non-pathogenicP. syringae(PG3a) appears to be associated with kiwifruit on a global scale. The presence of PG3a on kiwifruit has significant effects on the outcome of infection byP. syringaepv.actinidiae. This emphasises the value of studying the effect of co-occurring bacteria on pathogen-plant interactions.

scholarly journals Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato

2020 ◽  
Author(s):  
Eric C. Holmes ◽  
Yun-Chu Chen ◽  
Mary Beth Mudgett ◽  
Elizabeth S. Sattely
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Bacterial Pathogen ◽  
Defense Responses ◽  
Pipecolic Acid ◽  
Systemic Resistance ◽  
Biosynthetic Genes ◽  
Solanaceous Plants

AbstractSystemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in the response have not yet been elucidated. Here we report that Arabidopsis thaliana UGT76B1 can generate glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants do not accumulate NHP-Glc and accumulate less glycosylated salicylic acid (SA-Glc) than wild type plants. The metabolic changes in ugt76b1 mutant plants are accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of SAR molecules NHP and SA by UGT76B1 plays an important role in defense modulation. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato increases NHP-Glc production and reduces NHP accumulation in local tissue, and abolishes the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

scholarly journals Proteolytic Pathways of Activation and Degradation of a Bacterial Phospholipase C during Intracellular Infection by Listeria monocytogenes

1997 ◽  
Vol 137 (6) ◽  
pp. 1381-1392 ◽  
Author(s):  
Hélène Marquis ◽  
Howard Goldfine ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Pathogen ◽  
Specific Inhibitor ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Infected Cells ◽  
Extracellular Environment ◽  
Cell Spread ◽  
Proteolytic Pathways

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1–positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.

scholarly journals Antiviral Activity of Vacuolar ATPase Blocker Diphyllin against SARS-CoV-2

2021 ◽  
Vol 9 (3) ◽  
pp. 471
Author(s):  
Michal Stefanik ◽  
Petra Strakova ◽  
Jan Haviernik ◽  
Andrew D. Miller ◽  
Daniel Ruzek ◽  
...  
Keyword(s):  
Colorectal Adenocarcinoma ◽  
Vero Cells ◽  
Early Step ◽  
Endosomal Membranes ◽  
Infected Cells ◽  
Ph Dependent ◽  
Viral Envelopes ◽  
Vacuolar Atpases ◽  
Related Compounds

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.

scholarly journals CRK2 and C-terminal phosphorylation of NADPH oxidase RBOHD regulate ROS production in Arabidopsis

2019 ◽  
Author(s):  
Sachie Kimura ◽  
Kerri Hunter ◽  
Lauri Vaahtera ◽  
Huy Cuong Tran ◽  
Matteo Citterico ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Pseudomonas Syringae ◽  
Bacterial Pathogen ◽  
Terminal Region ◽  
Ros Production ◽  
Nadph Oxidases ◽  
C Terminus ◽  
Ros Burst ◽  
Eukaryotic Organisms

ABSTRACTReactive oxygen species (ROS) are important messengers in eukaryotic organisms and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. Functional CRK2 is required for the full elicitor-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment and substitution of S703 with alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating MAMP-triggered ROS production.One-sentence summaryCRK2 associates with and activates RBOHD to trigger MAMP-induced ROS production and reveals a novel regulatory mechanism for plant NADPH oxidases through phosphorylation of the C-terminus.

scholarly journals Arabidopsis MAPKKK δ-1 is required for full immunity against bacterial and fungal infection

2019 ◽  
Vol 71 (6) ◽  
pp. 2085-2097 ◽  
Author(s):  
Tomoya Asano ◽  
Thi Hang-Ni Nguyen ◽  
Michiko Yasuda ◽  
Yasir Sidiq ◽  
Kohji Nishimura ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Fungal Infection ◽  
Pseudomonas Syringae ◽  
Fusarium Species ◽  
Bacterial Pathogen ◽  
Mitogen Activated Protein Kinase ◽  
Trichothecene Mycotoxin ◽  
Mitogen Activated Protein

Abstract The genome of Arabidopsis encodes more than 60 mitogen-activated protein kinase kinase (MAPKK) kinases (MAPKKKs); however, the functions of most MAPKKKs and their downstream MAPKKs are largely unknown. Here, MAPKKK δ-1 (MKD1), a novel Raf-like MAPKKK, was isolated from Arabidopsis as a subunit of a complex including the transcription factor AtNFXL1, which is involved in the trichothecene phytotoxin response and in disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A MKD1-dependent cascade positively regulates disease resistance against PstDC3000 and the trichothecene mycotoxin-producing fungal pathogen Fusarium sporotrichioides. MKD1 expression was induced by trichothecenes derived from Fusarium species. MKD1 directly interacted with MKK1 and MKK5 in vivo, and phosphorylated MKK1 and MKK5 in vitro. Correspondingly, mkk1 mutants and MKK5RNAi transgenic plants showed enhanced susceptibility to F. sporotrichioides. MKD1 was required for full activation of two MAPKs (MPK3 and MPK6) by the T-2 toxin and flg22. Finally, quantitative phosphoproteomics suggested that an MKD1-dependent cascade controlled phosphorylation of a disease resistance protein, SUMO, and a mycotoxin-detoxifying enzyme. Our findings suggest that the MKD1–MKK1/MKK5–MPK3/MPK6-dependent signaling cascade is involved in the full immune responses against both bacterial and fungal infection.

scholarly journals Phospholipase C δ1 requires a pleckstrin homology domain for interaction with the plasma membrane

1995 ◽  
Vol 312 (3) ◽  
pp. 661-666 ◽  
Author(s):  
H F Paterson ◽  
J W Savopoulos ◽  
O Perisic ◽  
R Cheung ◽  
M V Ellis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Living Cells ◽  
Pleckstrin Homology Domain ◽  
Deletion Mutants ◽  
Pleckstrin Homology ◽  
Structural Requirements ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Attachment

The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

scholarly journals Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity

2017 ◽  
Vol 114 (28) ◽  
pp. 7456-7461 ◽  
Author(s):  
Akira Mine ◽  
Matthias L. Berens ◽  
Tatsuya Nobori ◽  
Shajahan Anver ◽  
Kaori Fukumoto ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Signaling Pathways ◽  
Pseudomonas Syringae ◽  
Map Kinases ◽  
Bacterial Pathogen ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Signaling Activation ◽  
Ja Signaling

Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana. ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1, HAI2, and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

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