The role of abscisic acid in the defence response of tomato (Solanum lycopersicum) to the necrotrophic pathogens Botrytis cinerea and Erwinia chrysanthemi

In order to cope with the constant threat of a wide range of potentially harmful micro-organisms, plants have developed an impressive constitutive and inducible defensive machinery of enormous complexity to combat pathogen invasion. Plant hormones are not only important for controlling plant development, but are also essential to regulate plant responses to the environment. The plant hormones salicylic acid (SA), jasmonate (JA) and ethylene (ET) are classically associated with plant pathogen defence responses as their increase in concentrations upon pathogen recognition, which leads to the activation of specific signalling cascades and pathogen defence gene expression, is important for resistance. In contrast, the plant hormone abscisic acid (ABA) has a well-established function in activating plant responses to abiotic stresses (such as cold, drought and salinity) by regulating stomatal aperture and by activating stress-responsive genes, but the effect of ABA on plant-pathogen interactions has long been a neglected research topic. Although exceptions exist, basal or high ABA levels are in most cases associated with susceptibility, while reduction of plant ABA levels often leads to increased resistance. However, our knowledge on the mechanisms of ABA-induced susceptibility is still very scarce and fragmentary. In the present work, we have explored the influence of ABA on the defence responses of tomato towards biotic stress. We have shown that ABA deficiency in the sitiens tomato mutant results in increased resistance towards the necrotrophic fungus Botrytis cinerea and the necrotrophic bacterium Erwinia chrysanthemi, two pathogens for which the occurrence of resistance is very rare. Comparison of gene expression in sitiens and wild-type tomato with TOM1 microarrays revealed that defence-related transcript accumulation prior to infection is higher in sitiens than in wild type. Moreover, further elevation of defence gene expression after pathogen attack is also stronger in sitiens, both in number of genes and their expression levels. These results show that ABA-deficiency results in priming for pathogen defence. Annotation of the genes differentially regulated between in sitiens and wild type showed elevated expression levels in sitiens of genes encoding SA-inducible PR proteins and of genes involved in SA biosynthesis, which confirmed earlier findings that ABA deficiency leads to hyperinduction of SA-dependent defence responses. Thorough analysis of the defence reactions that were hyperactivated in sitiens upon inoculation with B. cinerea and E. chrysanthemi revealed an essential role for hydrogen peroxide accumulation in the defence response towards both pathogens. Compared to wild-type tomato, hydrogen peroxide accumulation was earlier and more extensive in sitiens. The necessity of this defence reaction in the establishment of resistance in sitiens was demonstrated by disruption of the early and strong hydrogen peroxide accumulation. Removal of hydrogen peroxide with the antioxidants catalase and ascorbate and blocking hydrogen peroxide production with diphenilene iodonium increased the levels of susceptibility in sitiens. Accumulation of reactive oxygen species (ROS), such as hydrogen peroxide, during pathogen attack is a well-known phenomenon and its role in arresting biotrophic pathogens is firmly established. However, ROS accumulation is supposed to have a negative effect on defence against necrotrophic pathogens such as B. cinerea, since elevation of in planta ROS levels sets of a hypersensitive response, leading to increased plant tissue colonisation by necrotrophic pathogens. Our results clearly oppose the generally accepted theorem that plant defence-related ROS formation aids necrotrophs in their pathogenicity, and show that a timely hyperproduction of hydrogen peroxide is efficient in protecting the sitiens tomato mutant against necrotrophic pathogen attack. Although ROS can have other roles in defence, such as functioning as a signalling intermediate, having a direct antimicrobial effect or leading to a hypersensitive response, we have demonstrated that hyperaccumulation of hydrogen peroxide in sitiens leads to cell wall fortification, which results in containment of E. chrysanthemi and B. cinerea. Hydrogen peroxide accumulation in sitiens was accompanied by earlier and increased activation of extracellular peroxidases and a strong histochemical-detected cell wall fortification at the site of pathogen attack. These findings were consistent with the immediate hydrogen peroxide-fuelled peroxidative cross-linking of structural cell wall proteins and peroxidative incorporation of phenolic compounds during an oxidative burst. Furthermore, analysis of the genes activated in sitiens during B. cinerea inoculation revealed an overrepresentation of genes involved in cell wall modification. Microscopical analysis of pathogen progress in sitiens leaf tissue showed that both E. chrysanthemi (visualised directly as intercellular micro-colonies) and B. cinerea (visualised indirectly as progress of pectin degradation) were arrested by hydrogen peroxide-induced cell wall fortification. The site of pathogen arrest, i.e. the site of cell wall fortification, differed for E. chrysanthemi and B. cinerea, which was at least partly due to the nature of each inoculation procedure. B. cinerea penetrates the adaxial leaf cuticula and outer epidermal cell wall and was blocked by wall fortifications of the anticlinal epidermal sitiens cells. The arrest of E. chrysanthemi, which is infiltrated in the leaf tissue, was located at the sites of sitiens cell wall fortification at the border of the infiltration zone. Deposition of callose at the site of pathogen entry was previously demonstrated to be influenced by ABA. We have shown that callose deposition after B. cinerea inoculation is weaker in sitiens compared to the wild type. Inhibition of callose synthesis with 2-deoxy-D-glucose did not affect resistance in sitiens, but caused additional susceptibility in wild type. These findings indicate that callose deposition is not part of sitiens defence responses that are effective in blocking B. cinerea and suggest that callose deposition only contributes to wild-type tomato basal resistance. There are strong indications that the rapid and powerful defence responses in sitiens are triggered by recognition of endogenous plant cell wall elicitors. Both E. chrysanthemi and B. cinerea trigger the same type of defence responses in sitiens, which conflicts with the involvement of pathogen-specific elicitors. In addition, both pathogens produce great amounts of cell wall-degrading enzymes (CWDEs) and rely largely on cell wall pectin decomposition for their virulence. We have also shown that type II secretion-negative E. chrysanthemi mutants, which are incapable of secreting CWDEs, fail to fully activate sitiens defence responses. Furthermore, defence responses are elicited by bacteria-free CDWE-containing E. chrysanthemi culture filtrate. Since we found no differences between sitiens and wild-type in the capacity to resist E. chrysanthemi cell wall degradation, we have hypothesised that changes in sitiens cell wall composition could be responsible for differences in the release of cell wall oligomers, which are known and potent elicitors of defence responses upon pathogenic cell wall degradation. Alternatively, enhanced sensing of these oligomers in sitiens could also be responsible for the faster activation of defence. We found support for both hypotheses, as immunolocalisation of different cell wall components demonstrated differences between sitiens and wild-type in pectin distribution and in the presence of arabinogalactan proteins, which are presumably involved in signalling at the cell surface. Further experimental data will be needed expand our knowledge sitiens defence elicitation. In conclusion, we have shown that ABA-deficiency in tomato results in priming for pathogen defence responses and that a rapid hyperinduction of ROS-dependent cell wall fortification is a powerful defence strategy to stop the necrotrophic pathogens B. cinerea and E. chrysanthemi

HSPB1 facilitates the formation of non-centrosomal microtubules

The remodeling capacity of microtubules (MT) is essential for their proper function. In mammals, MTs are predominantly formed at the centrosome, but can also originate from non-centrosomal sites, a process that is still poorly understood. We here show that the small heat shock protein HSPB1 plays a role in the control of non-centrosomal MT formation. The HSPB1 expression level regulates the balance between centrosomal and non-centrosomal MTs. The HSPB1 protein can be detected specifically at sites of de novo forming non-centrosomal MTs, while it is absent from the centrosomes. In addition, we show that HSPB1 binds preferentially to the lattice of newly formed MTs in vitro, suggesting that its function occurs by stabilizing MT seeds. Our findings open new avenues for the understanding of the role of HSPB1 in the development, maintenance and protection of cells with specialized non-centrosomal MT arrays

Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Neutrophil cell death plays a crucial role in neutrophil homeostasis and the resolution of inflammation. The superoxide-producing NADPH oxidase is involved in pathogen degradation and subsequent activation of cell death programs. Neutrophils from patients with chronic granulomatous disease, who have a deficient NADPH oxidase activity, have been demonstrated previously to have a prolonged lifespan, suggesting that a basal NADPH oxidase activity also regulates spontaneous neutrophil turnover. The NADPH oxidase inhibitor parabutoporin (PP) does delay spontaneous apoptosis, but this effect is completely independent of NADPH oxidase inhibition. Instead, the prosurvival effect of PP depends on activation of protein kinase B/Akt via lipid raft signaling. Disruption of lipid rafts abrogates the prosurvival effect without interfering with NADPH oxidase activity. Furthermore, we cannot detect a different rate of spontaneous apoptosis between normal and NADPH oxidase-deficient neutrophils, arguing against a role of NADPH oxidase in spontaneous neutrophil apoptosis

Small heat-shock protein HSPB1 mutants stabilize microtubules in Charcot-Marie-Tooth neuropathy

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations

Sensory-neuropathy-causing mutations in ATL3 cause aberrant ER membrane tethering

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects

Sensory neuropathy-causing mutations in ATL3 affect ER-mitochondria contact sites and impair axonal mitochondrial distribution

Axonopathies are neurodegenerative disorders caused by axonal degeneration, affecting predominantly the longest neurons. Several of these axonopathies are caused by genetic defects in proteins involved in the shaping and dynamics of the endoplasmic reticulum (ER); however, it is unclear how these defects impinge on neuronal survival. Given its central and widespread position within a cell, the ER is a pivotal player in inter-organelle communication. Here, we demonstrate that defects in the ER fusion protein ATL3, which were identified in patients suffering from hereditary sensory and autonomic neuropathy, result in an increased number of ER-mitochondria contact sites both in HeLa cells and in patient-derived fibroblasts. This increased contact is reflected in higher phospholipid metabolism, upregulated autophagy and augmented Ca2+ crosstalk between both organelles. Moreover, the mitochondria in these cells display lowered motility, and the number of axonal mitochondria in neurons expressing disease-causing mutations in ATL3 is strongly decreased. These results underscore the functional interdependence of subcellular organelles in health and disease and show that disorders caused by ER-shaping defects are more complex than previously assumed

Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury

The authors identify two subsets of peripheral nerve macrophages residing in the endoneurium and the epineurium and displaying a distinct transcriptome and response to injury. These cells lack the main microglia identity and have a distinct origin. While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relm alpha(+)Mgl1(+) snMacs in the epineurium and Relm alpha(-)Mgl1(-) snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages

Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78)

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C>T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with frontotemporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement

Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations

Background: The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer's disease (AD). Although the actual risk-increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the beta-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. Results: Three patient-specific CLU mutations in the beta-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. Conclusions: Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the alpha- and beta-chain of CLU