Cell Defense Responses Research Articles

The pepper GNA-related lectin and PAN domain protein gene, CaGLP1, is required for plant cell death and defense signaling during bacterial infection

Carbohydrate-binding proteins, commonly referred to as lectins or agglutinins, function in defense responses to microbial pathogens. Pepper (Capsicum annuum) GNA-related lectin and PAN-domain protein gene CaGLP1 was isolated and functionally characterized from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaGLP1 contained an amine-terminus prokaryotic membrane lipoprotein lipid attachment site, a Galanthus nivalis agglutinin (GNA)-related lectin domain responsible for the recognition of high-mannose N-glycans, and a carboxyl-terminus PAN/apple domain. RNA gel blot and immunoblot analyses determined that CaGLP1 was strongly induced in pepper by compatible and incompatible Xcv infection. CaGLP1 protein localized primarily to the plasma membrane and exhibited mannose-binding specificity. CaGLP1-silenced pepper plants were more susceptible to compatible or incompatible Xcv infection compared with that of non-silenced control plants. CaGLP1 silencing in pepper leaves did not accumulate H2O2 and induce cell death during incompatible Xcv infection. Defense-related CaDEF1 (defensin) gene expression was significantly reduced in CaGLP1-silenced pepper plants. CaGLP1-overexpression in Arabidopsis thaliana enhanced resistance to Pseudomonas syringae pv. tomato. Defense-related AtPDF1.2 expression was elevated in CaGLP1-overexpression lines. Together, these results suggest that CaGLP1 is required for plant cell death and defense responses through the reactive oxygen species burst and downstream defense-related gene expression in response to bacterial pathogen challenge.

Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone.

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MSE. The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied.

Remodelling of actin cytoskeleton in tomato cells in response to inoculation with a biocontrol strain of Fusarium oxysporum in comparison to a pathogenic strain

Knowing that actin microfilaments play a key role in the mobilization of the defence‐associated cellular responses, the aim of this study was to compare changes affecting the actin cytoskeleton in tomato cells after inoculation with germinated microconidia of a biocontrol (Fo47, Fom24) or a pathogenic (Fol8) strain of Fusarium oxysporum. Actin microfilaments were observed by labelling with TRITC‐phalloidin combined with fluorescence microscopy. Results showed that only tenuous changes in the actin cytoskeleton architecture occurred after inoculation with the biocontrol strains whereas the actin cytoskeleton was significantly altered after inoculation with the pathogenic one. In the two types of interaction, cell death occurs and can be considered as one key component of cell defence responses. A pharmacological approach using cytochalasins was chosen to determine whether the inhibition of actin polymerization differently affects the kinetics of tomato cell death. Data showed that cytochalasins reduced cell death induced by the biocontrol strain Fo47, and in contrast, increased cell death induced by the pathogenic strain Fol8, suggesting that the pathway leading to cell death differs in the protective and compatible interactions.

Pepper aldehyde dehydrogenase CaALDH1 interacts with Xanthomonas effector AvrBsT and promotes effector-triggered cell death and defence responses.

Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient co-expression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves. Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1 acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection. Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1 interacts with AvrBsT and promotes plant cell death and defence responses.

ER quality control components UGGT and STT3a are required for activation of defense responses in bir1-1.

The receptor-like kinase SUPPRESSOR OF BIR1, 1 (SOBIR1) functions as a critical regulator in plant immunity. It is required for activation of cell death and defense responses in Arabidopsis bak1-interacting receptor-like kinase 1,1 (bir1-1) mutant plants. Here we report that the ER quality control component UDP-glucose:glycoprotein glucosyltransferase (UGGT) is required for the biogenesis of SOBIR1 and mutations in UGGT suppress the spontaneous cell death and constitutive defense responses in bir1-1. Loss of function of STT3a, which encodes a subunit of the oligosaccharyltransferase complex, also suppresses the autoimmune phenotype in bir1-1. However, it has no effect on the accumulation of SOBIR1, suggesting that additional signaling components other than SOBIR1 may be regulated by ER quality control. Our study provides clear evidence that ER quality control play critical roles in regulating defense activation in bir1-1.

The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses.

The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.

The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice.

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.

Extending the Adaptive Network Nanomedicine Model for Homeopathic Medicines: Nanostructures as Salient Cell Danger Signals for Adaptation

This paper examines the growing evidence supporting the adaptive network nanomedicine model for Homeopathic Medicines (HMs) and their actions. Multiple laboratories have identified nanostructures in homeopathically-manufactured medicines at low and high potencies. Replicated studies in mainstream pharmaceutical research and in homeopathy have also demonstrated elevated levels of elemental silicon and/or bioactive silica Nanoparticles (NPs) released from glassware during agitation or multiple homeopathic succussions. The model suggests that (a) very low potency HMs are complex mixtures of bulk, micro- and nanoscale forms of the medicine’s natural source material made by prolonged mechanical grinding (trituration) in dry lactose; (b) low and higher potency liquid HMs made in glass containers are nanocomposite materials formed from source NPs, nanosilica-coated source NPs, adjuvant nanosilica and source-doped, coated, seeded or template nanosilica in colloidal solutions that can survive drying. We hypothesize that HMs include hybrid nanostructures of various sources, small sizes, shapes, surface defects, zeta potentials, and surface reactivity. HMs serves as individually-salient, sub toxic virus-like foreign danger signals. Nanosilica would help carry and amplify the fingerprint signal of co-occurring and adsorbed source material on its surfaces at higher liquid potencies. Cell Defense Response (CDR) network constituents that HMs modulate involve gene expression, cytokine release, cell signaling, and cell stress mediators. Once triggered, nonlinear endogenous amplification processes facilitate evolution of the therapeutic response over time.

닭의 성숙/미성숙란에서 RNA Sequencing을 이용한 유전자 발현 양상 고찰

Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, KoreaABSTRACT Chicken eggs undergo various physiological changes during egg maturation. To study genes associated with the egg maturation in pre-ovulation (immature) and post-ovulation (mature), we compared gene expression patterns between in the immature egg and mature egg using RNA sequencing data. Mature and immature eggs were obtained from a Heuksaek Jaerae-jong of Korean native chicken. Total RNAs obtained from the eggs were sequenced by Illumina HiSeq 2000 platform, and the generated sequence reads were mapped to Galgal4 reference sequence assembly using Tuxedo Protocol. From the comparison of the RNA sequencing data, 315 genes were differentially expressed between mature and immature eggs, and 46 genes were only detected in immature egg. Further gene ontology (GO) analysis was performed for the differentially expressed genes using DAVID, showing that 29 and 28 GO terms were independently clustered from mature and immature, respectively. From those clustered GO terms, genes related to germ cell development, sex differentiation and defense response to bacterium were mainly expressed in the immature egg, while genes related to regulation of apoptosis, steroid metabolic process and lipid homeostasis were mainly detected in the mature egg. Our results could contribute to understand egg maturation before and after ovulation, and develop genetic markers for improving egg quality and productivity.(Key words : egg maturation, RNA sequencing, gene ontology, differential gene expression)

Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

Pepper Mitochondrial FORMATE DEHYDROGENASE1 Regulates Cell Death and Defense Responses against Bacterial Pathogens

Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide. Here, we report the identification and characterization of pepper (Capsicum annuum) mitochondrial FDH1 as a positive regulator of cell death and defense responses. Transient expression of FDH1 caused hypersensitive response (HR)-like cell death in pepper and Nicotiana benthamiana leaves. The D-isomer -: specific 2-hydroxyacid dehydrogenase signatures of FDH1 were required for the induction of HR-like cell death and FDH activity. FDH1 contained a mitochondrial targeting sequence at the N-terminal region; however, mitochondrial localization of FDH1 was not essential for the induction of HR-like cell death and FDH activity. FDH1 silencing in pepper significantly attenuated the cell death response and salicylic acid levels but stimulated growth of Xanthomonas campestris pv vesicatoria. By contrast, transgenic Arabidopsis (Arabidopsis thaliana) overexpressing FDH1 exhibited greater resistance to Pseudomonas syringae pv tomato in a salicylic acid-dependent manner. Arabidopsis transfer DNA insertion mutant analysis indicated that AtFDH1 expression is required for basal defense and resistance gene-mediated resistance to P. syringae pv tomato infection. Taken together, these data suggest that FDH1 has an important role in HR-like cell death and defense responses to bacterial pathogens.

A genome-wide association study of the maize hypersensitive defense response identifies genes that cluster in related pathways.

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.

Plant defence suppression is mediated by a fungal sirtuin during rice infection by Magnaporthe oryzae.

Crop destruction by the hemibiotrophic rice pathogen Magnaporthe oryzae requires plant defence suppression to facilitate extensive biotrophic growth in host cells before the onset of necrosis. How this is achieved at the genetic level is not well understood. Here, we report that a M. oryzae sirtuin, MoSir2, plays an essential role in rice defence suppression and colonization by controlling superoxide dismutase (SOD) gene expression. Loss of MoSir2 function in Δsir2 strains did not affect appressorial function, but biotrophic growth in rice cells was attenuated. Compared to wild type, Δsir2 strains failed to neutralize plant-derived reactive oxygen species (ROS) and elicited robust defence responses in rice epidermal cells that included elevated pathogenesis-related gene expression and granular depositions. Deletion of a SOD-encoding gene under MoSir2 control generated Δsod1 deletion strains that mimicked Δsir2 for impaired rice defence suppression, confirming SOD activity as a downstream output of MoSir2. In addition, comparative protein acetylation studies and forward genetic analyses identified a JmjC domain-containing protein as a likely target of MoSir2, and a Δsir2 Δjmjc double mutant was restored for MoSOD1 expression and defence suppression in rice epidermal cells. Together, this work reveals MoSir2 and MoJmjC as novel regulators of early rice cell infection.

Positive and negative roles for soybean MPK6 in regulating defense responses.

It has been well established that MPK6 is a positive regulator of defense responses in model plants such as Arabidopsis and tobacco. However, the functional importance of soybean MPK6 in disease resistance has not been investigated. Here, we showed that silencing of GmMPK6 in soybean using virus-induced gene silencing mediated by Bean pod mottle virus (BPMV) caused stunted growth and spontaneous cell death on the leaves, a typical phenotype of activated defense responses. Consistent with this phenotype, expression of pathogenesis-related (PR) genes and the conjugated form of salicylic acid were significantly increased in GmMPK6-silenced plants. As expected, GmMPK6-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants, indicating a negative role of GmMPK6 in disease resistance. Interestingly, overexpression of GmMPK6, either transiently in Nicotiana benthamiana or stably in Arabidopsis, resulted in hypersensitive response (HR)-like cell death. The HR-like cell death was accompanied by increased PR gene expression, suggesting that GmMPK6, like its counterpart in other plant species, also plays a positive role in cell death induction and defense response. Using bimolecular fluorescence complementation analysis, we determined that GmMKK4 might function upstream of GmMPK6 and GmMKK4 could interact with GmMPK6 independent of its phosphorylation status. Taken together, our results indicate that GmMPK6 functions as both repressor and activator in defense responses of soybean.

Profiling the Secretome and Extracellular Proteome of the Potato Late Blight Pathogen Phytophthora infestans

Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.

Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses Xanthomonas AvrBsT-triggered cell death and defense responses.

Pepper CaMLO2 specifically interacts with CaCaM1 and translocates cytoplasmic CaCaM1 to the plasma membrane, leading to the suppression of Xanthomonas AvrBsT-triggered Ca (2+) influx, hypersensitive cell death and defense responses. Pathogen-induced cell death is closely linked with disease susceptibility and resistance in plants. Pepper (Capsicum annuum) mildew resistance locus O (CaMLO2) and calmodulin (CaCaM1) genes are required for disease-associated cell death and hypersensitive cell death, respectively. Here, we demonstrate that pathogen-responsive CaMLO2 interacts with CaCaM1 in yeast and in planta. Bimolecular fluorescence complementation and co-immunoprecipitation analyses confirm a specific interaction between CaMLO2 and CaCaM1 at the plasma membrane (PM) in plant cells. Subcellular localization analyses of CaCaM1 fused to green fluorescent protein reveals that treatment with Ca(2+) and co-expression with CaMLO2 induce translocation of cytosolic CaCaM1 to the PM where CaMLO2 is localized. Transient CaMLO2 expression negatively regulates CaCaM1 accumulation in Nicotiana benthamiana. Xanthomonas avrBsT-triggered Ca(2+) influx and hypersensitive cell death are disrupted by CaCaM1 and/or CaMLO2 expression. CaMLO2 silencing in pepper significantly enhances reactive oxygen species burst, cell death, and resistance responses to Xanthomonas campestris pv. vesicatoria Ds1 and Ds1 (avrBsT), which is accompanied by enhanced induction of CaCaM1, CaPR1 (PR-1), and CaPO2 (peroxidase). These results suggest that CaMLO2 interacts with CaCaM1 and suppresses AvrBsT-triggered cell death and defense responses.

Continuous stimulation of the plant immune system by the peptide elicitor PIP-1 is required for phytoalexin biosynthesis in tobacco cells.

The peptide elicitor PIP-1 (YGIHTH-nh2) induced various defense responses in tobacco cells. Types of defense responses induced by PIP-1 were different based on its concentration range: oxidative burst (an early response) was induced at low micromolar levels, but phytoalexin production (a late response) required about 10-50-fold higher concentrations than those required for oxidative burst. We assumed that rapid decreases in the PIP-1 concentration due to enzymatic hydrolysis in the culture media could cause this difference. To examine the potential impact of such degradation particularly on induction of phytoalexin biosynthesis, we designed a degradation-resistant analogue, MePIP-1, in which the amide bond between the fifth and sixth residues was N-methylated. MePIP-1 was considerably more stable than PIP-1 and induced significant phytoalexin production upon treatment at low micromolar levels. Further investigation of the mechanism of action of MePIP-1 showed a requirement of continuous elicitor stimulation for 3-6 h for the phytoalexin production, which is likely to be regulated by long-lasting MAP kinase activation.

Activity and isoenzyme composition of vacuolar peroxidase in the roots of red beet at different stages of development and upon changes in storage conditions

Activity and isoenzyme composition of phenol-dependent vacuolar peroxidase (PO) were examined at different stages of development of red beet (Beta vulgaris L.) roots. The enzyme activity was found to increase during growth and decrease during the period of root dormancy. The isoenzyme composition of PO was also altered; additional cationic and anionic isoforms were revealed at certain stages of growth and dormancy. The dormant roots are often subjected to stresses, such as water deficiency and pathogenesis. For this reason, the enzyme activity in beet roots showing obvious signs of shrivel and infection with pathogenic microorganisms was investigated. The activation of PO was observed in infected roots, whereas the increase in the number of cationic PO isoforms was noted in water-stressed roots. The pH optima of the enzyme were found to shift depending on the developmental stage and the nature of stress factors: during dormancy and under water stress the pH optima shifted towards the acidic values. The shift in pH optima occurred concurrently with the appearance of cationic inducible isoforms. The pH dependences of cationic PO isoforms (these POs were mainly associated with the tonoplast) showed that their activity was optimal at pH 4.0 and 5.0. The observed changes in activity and composition of isoenzymes suggest the active involvement of the vacuolar peroxidase in metabolic processes and cell defense responses in beet roots.

Long intergenic non-coding RNA HOTAIRM1 regulates cell cycle progression during myeloid maturation in NB4 human promyelocytic leukemia cells

HOTAIRM1 is a long intergenic non-coding RNA encoded in the human HOXA gene cluster, with gene expression highly specific for maturing myeloid cells. Knockdown of HOTAIRM1 in the NB4 acute promyelocytic leukemia cell line retarded all-trans retinoid acid (ATRA)-induced granulocytic differentiation, resulting in a significantly larger population of immature and proliferating cells that maintained cell cycle progression from G1 to S phases. Correspondingly, HOTAIRM1 knockdown resulted in retained expression of many otherwise ATRA-suppressed cell cycle and DNA replication genes, and abated ATRA induction of cell surface leukocyte activation, defense response, and other maturation-related genes. Resistance to ATRA-induced cell cycle arrest at the G1/S phase transition in knockdown cells was accompanied by retained expression of ITGA4 (CD49d) and decreased induction of ITGAX (CD11c). The coupling of cell cycle progression with temporal dynamics in the expression patterns of these integrin genes suggests a regulated switch to control the transit from the proliferative phase to granulocytic maturation. Furthermore, ITGAX was among a small number of genes showing perturbation in transcript levels upon HOTAIRM1 knockdown even without ATRA treatment, suggesting a direct pathway of regulation. These results indicate that HOTAIRM1 provides a regulatory link in myeloid maturation by modulating integrin-controlled cell cycle progression at the gene expression level.