Heat shock factor gene family in rice: genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses

Binding of heat shock factors (HSFs) with heat shock element sequence is critical for the transcriptional induction of heat shock genes. Rice genome sequence shows 26 OsHsf genes out of which 25 possess various important domains noted in HSFs i.e. DNA binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES) and AHA type activation domain. OsHsf entry LOC_Os06g226100 has the oligomerization domain but lacks the above other domains. Also, there are no ESTs or full-length cDNA noted for this entry in database. Expression profiling showed that 22 OsHsf genes are induced by high temperature. Induction of 10 and 14 OsHsf genes was also noted against low temperature stress and oxidative stress, respectively. All OsHsf genes induced by oxidative stress were also induced by high temperature. On the other hand, induction of 6 and 1 OsHsf genes was noted to be exclusive to high and low temperature stresses, respectively. Seven OsHsf genes showed induced expression in response to all the three stresses examined. While in silico promoter analysis showed that OsHsf genes contain upstream regulatory elements corresponding to different abiotic stresses, there was lack of correlation noted between the in silico profiling of the elements and their corresponding transcript expression patterns. Apart from stress inducibility, EST database suggests that various OsHsf genes are developmentally regulated in diverse tissue types

The Drosophila MAPK p38c Regulates Oxidative Stress and Lipid Homeostasis in the Intestine

The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. In this study, we have analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. We showed that p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora 15. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Duox is reduced in p38c mutant flies. Our genetic analysis shows that p38c functions in a pathway with Mekk1 and Mkk3 to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, our study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c

Infection-induced host translational blockage inhibits immune responses and epithelial renewal in the Drosophila gut

Typically, immune responses control the pathogen, while repair and stress pathways limit damage caused by pathogenesis. The relative contribution of damage to the outcome of pathogenesis and the mechanistic links between the immune and repair pathways are poorly understood. Here, we analyze how the entomopathogenic bacterium Pseudomonas entomophila induces irreversible damage to the Drosophila gut. We find that P. entomophila ingestion induces a global translational blockage that impairs both immune and repair programs in the fly gut. P. entomophila-induced translational inhibition is dependent on bacterial pore forming toxins and reactive oxygen species produced by the host in response to infection. Translational arrest is mediated through activation of the GCN2 kinase and inhibition of the TOR pathway as a consequence of host damage. Together, our study draws a model of pathogenesis in which bacterial inhibition of translation by excessive activation of stress responsive pathways inhibits both immune and regenerative epithelial responses

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis

Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK–STAT (Janus kinase–signal transducers and activators of transcription) and JNK (c-Jun NH2 terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity

Genes involved in antimicrobial response, stress response, and metabolism are differentially regulated in <i>p38c^7B1</i> flies.

<p>(<b>A</b>) Proportion of up-regulated (left) and down-regulated (right) genes in different Gene Ontology categories. (<b>B</b>) A selection of genes differentially regulated in <i>p38c^7B1</i> fly guts (fold change compared to wild-type). Gene categories were determined by GO analysis on DAVID. Genes also affected in the <i>atf3⁷⁶</i> mutant larvae as described in reference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004659#pgen.1004659-Rynes1" target="_blank">[33]</a> are highlighted with an #. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004659#pgen.1004659.s009" target="_blank">Table S1</a> for complete list of genes.</p

p38c and Atf3 function in a same pathway to control lipid homeostasis.

<p>(<b>A</b>) <i>p38c^7B1</i> and <i>Mekk1^Ur36</i> mutants accumulate lipids in regions of the midgut as observed by Nile Red. Enlarged lipid droplets were observed in the gut of <i>p38c</i> but not the wild-type or <i>p38c;P[p38c]</i> flies. <i>Atf-2</i> mutants showed a modest accumulation of neutral lipids as observed by Nile Red, when compared to the wild-type. (<b>B</b>) A lineage tracing system using <i>esg^tsF/O</i> to silence <i>p38c</i> by RNAi in specific cells. Cells with reduced <i>p38c</i> expression (green) had increased lipid accumulation (yellow) relative to surrounding enterocytes. (<b>C</b>) RT-qPCR analysis of <i>Atf3</i> expression in 3–5 day old adult female fly intestines. Data are the mean of three repeats and error bars show standard error. * = p<0.05 determined by Student's <i>t</i> test. Genotypes are indicated on the x-axis, WT: <i>w¹¹¹⁸</i>. (<b>D</b>) The over-expression of <i>atf3</i> in <i>p38c</i> mutant flies (genotype: <i>NP1-GAL4;UAS-ATF3,p38c^7B1/p38c^7B1</i>) restores a wild-type level of lipid in the gut.</p

A Mekk1-Mkk3-P38c-Atf-2 pathway regulate <i>Duox</i> expression.

<p>(<b>A</b>) The induction of <i>Duox</i> upon <i>P. entomophila</i> infection is reduced in <i>p38c^7B1</i> mutant flies. (<b>B</b>) Over-expressing <i>p38c</i> in the gut induced a higher level of <i>Duox</i> expression in the absence of infection. WT: <i>NP1-GAL4; +</i>. (<b>C</b>) Mekk1 and Mkk3 but not PGRP-LC regulates expression of <i>Duox</i> upon infection. Genotype: <i>w¹¹¹⁸ (wild-type)</i>, <i>MEKK1^Ur36</i>, <i>PGRP-LC^E12</i>, <i>[PGRP-LC]; PGRP-LC^E12</i> (a wild-type line with the same genetic background as <i>PGRP-LC^E12</i>), and <i>NP1-GAL4;UAS-Mkk3IR</i>. (<b>D</b>) <i>Duox</i> expression was reduced in <i>Atf-2</i> flies upon infection. In (<b>A</b>, <b>C</b> and <b>D</b>) <i>Duox</i> expression was monitored by RT-qPCR performed with total RNA extracts from guts collected 2 h after <i>P. entomophila</i> infection. (<b>E</b>) Quantification of bacterial-induced ROS (H₂O₂) generation in gut extracts from adult female flies collected at 45 minutes post-infection with <i>P. entomophila</i> using Amplex Red reagent (Invitrogen). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004659#s4" target="_blank">methods</a> for details. Mean values of three experiments (N = 10 guts each) ± SE are shown (<b>F</b>) Atf-2 is phosphorylated in wild-type but not in <i>p38c^7B1</i> flies. The level of phosphorylation was only partially rescue in <i>p38c^7B1, P[p38c]</i>. Western blot were performed on gut collected 4 h following oral infection with <i>P. entomophila</i>. The total levels of Atf-2 remain unchanged in all genotypes with or without infection. (<b>G</b>) <i>atf-2</i> deficient flies shows that an increase survival rate compared to wild-type orally infected with <i>P. entomophila</i>. Mean values of at least three experiments (N = 10 to 20 flies each) ± SE are shown. * p<0.05;, and NS: non-significant as determined by Student's <i>t</i> test. Kaplan-Meier log-rank test was used in (<b>G</b>) to determine statistical significance *** p<0.001.</p

<i>p38c</i> is induced in the intestine following oral bacterial infection.

<p>(<b>A</b>) Data from Flyatlas showed an enrichment of p38c in the midgut <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004659#pgen.1004659-Ha3" target="_blank">[30]</a>. Expression is shown as a ratio of mRNA enrichment for each gene in each tissue to the average mRNA enrichment for all the tissues. T ganglion: thoracic abdominal ganglion (<b>B</b>) <i>p38c</i> expression is the most induced <i>p38</i> gene in the midgut upon oral bacterial infection. The induction of the <i>p38</i> genes of <i>Drosophila</i> was monitored on gut RNA extracts of wild-type flies using RT-qPCR. Guts were collected at different time points (2, 4, 6 and 16 h) following oral infection with <i>Ecc15</i> and <i>P. entomophila (Pe)</i>. The level of induction of both <i>p38a</i> and <i>p38c</i> was higher following <i>P. entomophila</i> infection and peaked at 6 h post-infection. (<b>C</b>) Confoncal images of the anterior midgut stained with an anti-p38c serum of female flies either unchallenged (<b>C1</b>) collected 16 h after <i>P. entomophila</i> infection (<b>C2</b>). Insets show higher magnification. p38c is shown in red, nuclei are in blue. UC: unchallenged control. Diffuse or punctate signals corresponding to p38c protein were observed in the cytoplasm of enterocyte.</p