Function of endoplasmic reticulum calcium ATPase in innate immunity‐mediated programmed cell death

Programmed cell death (PCD) initiated at the pathogen‐infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER‐localized type IIB Ca²⁺‐ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N‐ and fungal‐immune receptor Cf9‐mediated PCD, as well as non‐host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein‐induced cell death. The accelerated PCD rescues loss‐of‐resistance phenotype of Rar1, HSP90‐silenced plants, but not SGT1‐silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N‐immune receptor‐mediated PCD. Our results indicate that ER‐Ca²⁺‐ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.     

Over‐expression of HSP70 attenuates caspase‐dependent and caspase‐independent pathways and inhibits neuronal apoptosis

HSP70 is a member of the family of heat‐shock proteins that are known to be up‐regulated in neurons following injury and/or stress. HSP70 over‐expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over‐expression by transfection with HSP70‐expression plasmids in primary cortical neurons and the SH‐SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2‐ceramide, and β‐Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease‐activating factor 1, as well as apoptosis‐inducing factor, key molecules involved in development of caspase‐dependent and caspase‐independent PCD, respectively. Markers of caspase‐dependent PCD, including active caspase‐3, caspase‐9, and cleaved PARP were attenuated in neurons over‐expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase‐dependent and caspase‐independent PCD pathways.     

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF‐1 signalling pathway, IGF‐I and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) were used to treat MCF‐7 and MDA‐MB‐231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide‐treated breast cancer cells. PI3K inhibitor and IGF‐I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide‐treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Involvement of ethylene and nitric oxide in cell death in mastoparan‐treated unicellular alga Chlamydomonas reinhardtii

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)‐induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene‐ and NO‐interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP‐induced cell death. By employing a carbon dioxide laser‐based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)‐based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP‐induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP‐induced cell death.     

Platycodin D suppresses cisplatin‐induced cytotoxicity by suppressing ROS‐mediated oxidative damage,apoptosis, and inflammation in HEK‐293 cells

Cisplatin, a proven effective chemotherapeutic agent, has been used clinically to treat malignant solid tumors, whereas its clinical use is limited by serious side effect including nephrotoxicity. Platycodin D (PD), the major and marked saponin isolated from Platycodon grandiflorum, possesses many pharmacological effects. In this study, we evaluated its protective effect against cisplatin‐induced human embryonic kidney 293 (HEK‐293) cells injury and elucidated the related mechanisms. Our results showed that PD (0.25, 0.5, and 1 μM) can dose‐dependently alleviate oxidative stress by decreasing malondialdehyde and reactive oxygen species, while increasing the levels of glutathione, superoxide dismutase, and catalase. Moreover, the elevation of apoptosis including Bax, Bad, cleaved caspase‐3,‐9, and decreased protein levels of Bcl‐2, Bcl‐XL induced by cisplatin were reversed after PD treatment. Importantly, PD pretreatment can also regulate PI3K/Akt and ERK/JNK/p38 signaling pathways. Furthermore, PD was found to reduce NF‐κB‐mediated inflammatory relative proteins. Our finding indicated that PD exerted significant effects on cisplatin induced oxidative stress, apoptosis and inflammatory, which will provide evidence for the development of PD to attenuate cisplatin‐induced nephrotoxicity.     

A chemical–genetic interaction map of small molecules using high‐throughput imaging in cancer cells

Small molecules often affect multiple targets, elicit off‐target effects, and induce genotype‐specific responses. Chemical genetics, the mapping of the genotype dependence of a small molecule's effects across a broad spectrum of phenotypes can identify novel mechanisms of action. It can also reveal unanticipated effects and could thereby reduce high attrition rates of small molecule development pipelines. Here, we used high‐content screening and image analysis to measure effects of 1,280 pharmacologically active compounds on complex phenotypes in isogenic cancer cell lines which harbor activating or inactivating mutations in key oncogenic signaling pathways. Using multiparametric chemical–genetic interaction analysis, we observed phenotypic gene–drug interactions for more than 193 compounds, with many affecting phenotypes other than cell growth. We created a resource termed the Pharmacogenetic Phenome Compendium (PGPC), which enables exploration of drug mode of action, detection of potential off‐target effects, and the generation of hypotheses on drug combinations and synergism. For example, we demonstrate that MEK inhibitors amplify the viability effect of the clinically used anti‐alcoholism drug disulfiram and show that the EGFR inhibitor tyrphostin AG555 has off‐target activity on the proteasome. Taken together, this study demonstrates how combining multiparametric phenotyping in different genetic backgrounds can be used to predict additional mechanisms of action and to reposition clinically used drugs.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

Exercise reverses age‐related vulnerability of the retina to injury by preventing complement‐mediated synapse elimination via a BDNF‐dependent pathway

Retinal ganglion cells (RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise‐induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain‐derived neurotrophic factor (BDNF) and AMP‐activated protein kinase (AMPK). In middle‐aged (12 months old) C57BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra‐ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock‐down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement‐mediated elimination of synapses.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

Selenium induces a multi‐targeted cell death process in addition to ROS formation

Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi‐target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium‐mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno‐DL‐cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC₅₀ 5 μM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis‐like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis‐like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno‐DL‐cystine treatment at an IC₅₀ concentration of 100 μM induced morphologically two distinct different types of cell death, one with apoptosis‐like phenotype, while the other was reminiscent of paraptosis‐like cell death, characterized by induction of unfolded protein response, ER‐stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species‐specific usage of selenium in the treatment of cancers.     

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐positive cells both in inactive and feeding animals. The DA‐induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto‐c (cytochrome c) translocation into the cytosol and methyl‐β‐cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto‐c was under the control of Bcl‐2 and Bad. DA also increased the active caspase‐3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter‐mediated pathway that regulates the PCD of secretory cells in a mitochondrial‐caspase‐dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto‐c, ceramide, Bcl‐2 proteins and caspase‐3, but not caspase‐8, was demonstrated in cells involved in the DA‐induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.     

Aging differentially modulates the Wnt pro‐survival signalling pathways in vascular smooth muscle cells

We previously reported pro‐survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1‐inducible signalling pathway protein‐1 (WISP‐1) consequent to β‐catenin/CREB‐dependent, TCF‐independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP‐1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro‐survival signalling and demonstrate the differential effect of age on Wnt3a‐ and Wnt5a‐mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co‐located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased β‐catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro‐survival factor WISP‐2 in a TCF‐dependent manner. Functionally, β‐catenin/TCF inhibition or WISP‐2 neutralization significantly impaired Wnt3a‐mediated VSMC survival. WISP‐2 was upregulated in human atherosclerosis and partly co‐localized with Wnt3a. The pro‐survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18–20 month) mice, whereas Wnt5a‐mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced β‐catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP‐1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro‐survival Wnt3a signalling involves β‐catenin/TCF and WISP‐2. While Wnt3a‐mediated survival was unchanged with age, Wnt5a‐mediated survival was lost due to impaired CREB activation and WISP‐1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis.     

Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.     

Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Bax assembles into large ring‐like structures remodeling the mitochondrial outer membrane in apoptosis

The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells.     

Cell‐wall invertases,key enzymes in the modulation of plant metabolism during defence responses

Most plant–pathogen interactions do not result in pathogenesis because of pre‐formed defensive plant barriers or pathogen‐triggered activation of effective plant immune responses. The mounting of defence reactions is accompanied by a profound modulation of plant metabolism. Common metabolic changes are the repression of photosynthesis, the increase in heterotrophic metabolism and the synthesis of secondary metabolites. This enhanced metabolic activity is accompanied by the reduced export of sucrose or enhanced import of hexoses at the site of infection, which is mediated by an induced activity of cell‐wall invertase (Cw‐Inv). Cw‐Inv cleaves sucrose, the major transport sugar in plants, irreversibly yielding glucose and fructose, which can be taken up by plant cells via hexose transporters. These hexose sugars not only function in metabolism, but also act as signalling molecules. The picture of Cw‐Inv regulation in plant–pathogen interactions has recently been broadened and is discussed in this review. An interesting emerging feature is the link between Cw‐Inv and the circadian clock and new modes of Cw‐Inv regulation at the post‐translational level.