In the scenario of global warming and climate change, heat stress is a serious threat to crop production worldwide. Being sessile, plants cannot escape from heat. Plants have developed various adaptive mechanisms to survive heat stress. Several studies have focused on diversity of heat tolerance levels in divergent Arabidopsis thaliana (A. thaliana) ecotypes, but comprehensive genome scale understanding of heat stress response in plants is still lacking. Here we report the genome scale transcript responses to heat stress of 10 A. thaliana ecotypes (Col, Ler, C24, Cvi, Kas1, An1, Sha, Kyo2, Eri, and Kond) originated from different geographical locations. During the experiment, A. thaliana plants were subjected to heat stress (38°C) and transcript responses were monitored using Arabidopsis NimbleGen ATH6 microarrays. The responses of A. thaliana ecotypes exhibited considerable variation in the transcript abundance levels. In total, 3644 transcripts were significantly heat regulated (p < 0.01) in the 10 ecotypes, including 244 transcription factors and 203 transposable elements. By employing a systems genetics approach- Network Component Analysis (NCA), we have constructed an in silico transcript regulatory network model for 35 heat responsive transcription factors during cellular responses to heat stress in A. thaliana. The computed activities of the 35 transcription factors showed ecotype specific responses to the heat treatment.

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.

The systematic comparison of transcriptional responses of organisms is a powerful tool in functional genomics. For example, mutants may be characterized by comparing their transcript profiles to those obtained in other experiments querying the effects on gene expression of many experimental factors including treatments, mutations and pathogen infections. Similarly, drugs may be discovered by the relationship between the transcript profiles effectuated or impacted by a candidate drug and by the target disease. The integration of such data enables systems biology to predict the interplay between experimental factors affecting a biological system. Unfortunately, direct comparisons of gene expression profiles obtained in independent, publicly available microarray experiments are typically compromised by substantial, experiment-specific biases. Here we suggest a novel yet conceptually simple approach for deriving 'Functional Association(s) by Response Overlap' (FARO) between microarray gene expression studies. The transcriptional response is defined by the set of differentially expressed genes independent from the magnitude or direction of the change. This approach overcomes the limited comparability between studies that is typical for methods that rely on correlation in gene expression. We apply FARO to a compendium of 242 diverse Arabidopsis microarray experimental factors, including phyto-hormones, stresses and pathogens, growth conditions/stages, tissue types and mutants. We also use FARO to confirm and further delineate the functions of Arabidopsis MAP kinase 4 in disease and stress responses. Furthermore, we find that a large, well-defined set of genes responds in opposing directions to different stress conditions and predict the effects of different stress combinations. This demonstrates the usefulness of our approach for exploiting public microarray data to derive biologically meaningful associations between experimental factors. Finally, our results indicate that FARO is more powerful in associating mutants in common pathways than existing methods such as co-expression analysis.

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

Multi-layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de-repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses.

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers worldwide. In search for new NSCLC treatment options, we screened a cationic amphiphilic drug (CAD) library for cytotoxicity against NSCLC cells and identified several CAD antihistamines as inducers of lysosomal cell death. We then performed a cohort study on the effect of CAD antihistamine use on mortality of patients diagnosed with non-localized cancer in Denmark between 1995 and 2011. The use of the most commonly prescribed CAD antihistamine, loratadine, was associated with significantly reduced all-cause mortality among patients with non-localized NSCLC or any non-localized cancer when compared with use of non-CAD antihistamines and adjusted for potential confounders. Of the less frequently described CAD antihistamines, astemizole showed a similar significant association with reduced mortality as loratadine among patients with any non-localized cancer, and ebastine use showed a similar tendency. The association between CAD antihistamine use and reduced mortality was stronger among patients with records of concurrent chemotherapy than among those without such records. In line with this, sub-micromolar concentrations of loratadine, astemizole and ebastine sensitized NSCLC cells to chemotherapy and reverted multidrug resistance in NSCLC, breast and prostate cancer cells. Thus, CAD antihistamines may improve the efficacy of cancer chemotherapy.

In plant innate immunity, the surface-exposed leucine-rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen-associated molecular patterns EF-Tu and flagellin, respectively. We identified the Arabidopsis stromal-derived factor-2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER-quality control (ER-QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER-QC components by EFR and FLS2 could be linked to N-glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co-translational N-glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2-ERdj3B-BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER-QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER-QC and N-glycosylation components by two closely related receptors.

Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins.

Programmed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins.

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Heat Shock Proteins (HSPs) maintain cellular homeostasis under stress. HSP70 represents a major stress-inducible family member and has been identified as a druggable target in inherited cholesterol-sphingolipid storage diseases. We investigated if HSP70 modulates cholesterol accumulation in more common conditions related to atherogenesis.

Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy.

DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity. However, other immune responses may suppress DNA damage to maintain host genome integrity. Here, we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding, Leucine-rich-repeat immune Receptors (NLRs). SNI1 (suppressor of npr1-1, inducible 1), a subunit of the structural maintenance of chromosome (SMC) 5/6 complex, was reported to be a negative regulator of systemic acquired resistance (SAR) and to be necessary for controlling DNA damage. We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1. Similar to sni1, elevated DNA damage is seen in other autoimmune mutants with cell death lesions, including camta3, pub13 and vad1, but not in dnd1, an autoimmune mutant with no visible cell death. We find that as in sni1, DNA damage in camta3 is EDS1-dependent, but that it is also NLR-dependent. Using the NLR RPM1 as a model, we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors. We also find that the expression of DNA damage repair (DDR) genes in mutants with cell death lesions is down regulated, suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair. Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation.

To establish infection, pathogens deploy effectors to modify or remove host proteins. Plant immune receptors with nucleotide-binding, leucine-rich repeat domains (NLRs) detect these modifications and trigger immunity. Plant NLRs thus guard host "guardees." A corollary is that autoimmunity may result from inappropriate NLR activation because mutations in plant guardees could trigger corresponding NLR guards. To explore these hypotheses, we expressed 108 dominant-negative (DN) Arabidopsis NLRs in various lesion mimic mutants, including camta3, which exhibits autoimmunity. CAMTA3 was previously described as a negative regulator of immunity, and we find that autoimmunity in camta3 is fully suppressed by expressing DNs of two NLRs, DSC1 and DSC2. Additionally, expression of either NLR triggers cell death that can be suppressed by CAMTA3 expression. These findings support a model in which DSC1 and DSC2 guard CAMTA3, and they suggest that other negative regulators of immunity may similarly represent guardees.

Niemann-Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease associated with neurovisceral manifestations resulting from lysosomal dysfunction and aberrant lipid accumulation. A multicentre, prospective observational study (Clinical Trials.gov ID: NCT02435030) of individuals with genetically confirmed NPC1 or NPC2 receiving routine clinical care was conducted, to prospectively characterize and measure NPC disease progression and to investigate potential NPC-related biomarkers versus healthy individuals. Progression was measured using the abbreviated 5-domain NPC Clinical Severity Scale (NPCCSS), 17-domain NPCCSS and NPC clinical database (NPC-cdb) score. Cholesterol esterification and heat shock protein 70 (HSP70) levels were assessed from peripheral blood mononuclear cells (PBMCs), cholestane-3β,5α-,6β-triol (cholestane-triol) from serum, and unesterified cholesterol from both PBMCs and skin biopsy samples. The inter- and intra-rater reliability of the 5-domain NPCCSS was assessed by 13 expert clinicians' rating of four participants via video recordings, repeated after ≥ 3 weeks. Intraclass correlation coefficients (ICCs) were calculated.

The insectivorous Venus flytrap (Dionaea muscipula) is renowned from Darwin's studies of plant carnivory and the origins of species. To provide tools to analyze the evolution and functional genomics of D. muscipula, we sequenced a normalized cDNA library synthesized from mRNA isolated from D. muscipula flowers and traps. Using the Oases transcriptome assembler 79,165,657 quality trimmed reads were assembled into 80,806 cDNA contigs, with an average length of 679 bp and an N50 length of 1,051 bp. A total of 17,047 unique proteins were identified, and assigned to Gene Ontology (GO) and classified into functional categories. A total of 15,547 full-length cDNA sequences were identified, from which open reading frames were detected in 10,941. Comparative GO analyses revealed that D. muscipula is highly represented in molecular functions related to catalytic, antioxidant, and electron carrier activities. Also, using a single copy sequence PCR-based method, we estimated that the genome size of D. muscipula is approx. 3 Gb. Our genome size estimate and transcriptome analyses will contribute to future research on this fascinating, monotypic species and its heterotrophic adaptations.

Low temperature leads to major crop losses every year. Although several studies have been conducted focusing on diversity of cold tolerance level in multiple phenotypically divergent Arabidopsis thaliana (A. thaliana) ecotypes, genome-scale molecular understanding is still lacking.

Autophagy has been implicated as a prosurvival mechanism to restrict programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. This model is based on the observation that HR lesions spread in plants with reduced autophagy gene expression. Here, we examined receptor-mediated HR PCD responses in autophagy-deficient Arabidopsis knockout mutants (atg), and show that infection-induced lesions are contained in atg mutants. We also provide evidence that HR cell death initiated via Toll/Interleukin-1 (TIR)-type immune receptors through the defense regulator EDS1 is suppressed in atg mutants. Furthermore, we demonstrate that PCD triggered by coiled-coil (CC)-type immune receptors via NDR1 is either autophagy-independent or engages autophagic components with cathepsins and other unidentified cell death mediators. Thus, autophagic cell death contributes to HR PCD and can function in parallel with other prodeath pathways.