Transcriptional insights of citrus defense response against Diaporthe citri.

Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6'H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism.

Testing Emotional Vulnerability to Threat in Adults Using a Virtual Reality Paradigm of Fear Associated With Autonomic Variables.

Fear and anxiety are generally assessed as responses of prey to high or low levels of threatening environments, fear-conditioned or unconditioned stimuli, or the intensity and distance between predator and prey. Depending on whether a threat is close to or distant from the individual, the individual exhibits specific behaviors, such as being quiet (freezing in animals) if the threat is distant or fleeing if the threat is close. In a seminal paper in 2007, Dean Mobbs developed an active prevention virtual reality paradigm (VRP) to study a threat's spatial imminence using finger shocks. In the present study, we used a modified VRP with a distinctive feature, namely a dynamic threat-of-loud noise paradigm. The results showed a significant reduction in the number of times the subjects were captured in the high predator phase (85 dB) vs. control phases, suggesting that the participants were motivated to avoid the high predator. Concomitant with avoidance behavior, a decrease in respiratory rate and an increase in heart rate characterized the defense reaction. These results demonstrate behavioral and autonomic effects of threat intensity in volunteers during a VRP, revealing a profile of defense reaction that reflects the individual emotional susceptibility to the development of anxiety.

Role of Benzoic Acid and Lettucenin A in the Defense Response of Lettuce against Soil-Borne Pathogens.

Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.

The Invasion and Encapsulation of the Entomopathogenic Nematode, Steinernema abbasi, in Aedes albopictus (Diptera: Culicidae) Larvae.

The Asian tiger mosquito, Aedes albopictus, is of crucial concern to the public and veterinary health because of its vector role in transmission of several mosquito-borne diseases. Over the past decades, entomopathogenic nematodes (EPNs) have been used to control important agricultural insect pests and are considered to be effective against mosquitoes as well. The objectives of this study were to investigate the mosquitocidal effects of Steinernema abbasi to Ae. albopictus and the encapsulation processes of invading nematodes in the mosquito host. In this study, we found that S. abbasi was pathogenic to 3rd and 4th instar larvae of Ae. albopictus by entering the hemocoel of the 3rd and 4th instar larvae mainly through mouth and gastric caecum or by penetrating pupae through the intersegmental membrane or trumpet. The mosquito larvae infected with a single nematode caused a high mortality. Although EPNs in the hemocoel of mosquitoes were melanized and encapsulated, most Ae. albopictus larvae failed to survive after infection with S. abbasi. Overall, we demonstrated that S. abbasi is pathogenic to Ae. albopictus larvae, suggesting that this S. abbasi isolate has potential as a biocontrol agent for managing this vector mosquito.

Ventromedial medullary pathway mediating cardiac responses evoked from periaqueductal gray.

Periaqueductal gray (PAG) is a midbrain region that projects to areas controlling behavioral and autonomic outputs and is involved in the behavioral and physiological components of defense reactions. Since Raphe Pallidus (RPa) is a medial medullary region comprising sympathetic premotor neurons governing heart function, it is worth considering the PAG-RPa path. We assessed: i) whether PAG projects to RPa; ii) the amplitude of cardiac responses evoked from PAG; iii) whether cardiovascular responses evoked from PAG rely on RPa. Experiments conducted in Wistar rats (±300 g) were approved by Ethics Committee CEUA-UFG (092/18). Firstly, (n = 3), monosynaptic retrograde tracer Retrobeads was injected into RPa; PAG slices were analyzed. Other two groups (n = 6 each) were anesthetized with urethane (1.4 g/kg) and chloralose (120 mg/kg) and underwent craniotomy, tracheostomy, catheterization of femoral artery and vein and of cardiac left ventricle. In one group, we injected the GABAA receptor antagonist, bicuculline methiodide (BMI - 40 pmol/100 nL) into lateral/dorsolateral PAG. Another group was injected (100 nL) with the GABAA receptor agonist muscimol (20 mM) into RPa, 20 min before BMI into PAG. The results were: i) retrogradely labelled neurons were found in PAG; ii) PAG activation by BMI caused positive chronotropism and inotropism, which were accompanied by afterload increases; iii) RPa inhibition with Muscimol reduced heart rate, arterial and ventricular pressures; iv) the subsequent PAG activation still increased arterial pressure, cardiac chronotropy and inotropy, but these responses were significantly attenuated. In conclusion, PAG activation increases cardiac chronotropy and inotropy, and these responses seem to rely on a direct pathway reaching ventromedial medullary RPa neurons.

ROS Synthesis and Accumulation during Wound-induced Sesquiterpene Biosynthesis in Aquilaria sinensis Callus / 中国药学杂志

OBJECTIVE: To explore the response intensity of reactive oxygen species(ROS) components including H2O2, O-2 and OH- in the course of wound-induced sesquiterpenes formation in Aquilaria sinensis callus. METHODS: The synthesis of sesquiterpenes in wounded callus was analyzed by using GG-MS technique. Total ROS distribution in cells was observed by fluorescence microscope after H2DCF fluorescent probe staining. The H2O2, O-2 and OH- contents were measured according to biochemical reaction principle. RESULTS: Two sesquitterpenes were synthesized and accumulated in the wounded cells. As a signal molecule, ROS burst and accumulated in the process of wound-induced sesquiterpenes formation, DCF fluorescence was observed to increase gradually. DCF fluorescence reached peak at 3 d and then began to decrease. The response of H2O2 and O-2 to injury took place earlier than those of OH-, H2O2 and O-2 reached the maximum value at 3 d and then decreased, while OH- reached the maximum value at 5 d. CONCLUSION: All ROS components respond to injury stimulation as signaling molecules, and the difference in response intensity of each component can synergistically regulate defense response to promote the synthesis of sesquiterpenes in A.sinensis callus.

Identification of tomato miRNAs responsive to root colonization by endophytic Pochonia chlamydosporia.

The molecular mechanisms active during the endophytic phase of the fungus Pochonia chlamydosporia are still poorly understood. In particular, few data are available on the links between the endophyte and the root response, as modulated by noncoding small RNAs. In this study, we describe the microRNAs (miRNAs) that are differentially expressed (DE) in the roots of tomato, colonized by P. chlamydosporia. A genome-wide NGS expression profiling of small RNAs in roots, either colonized or not by the fungus, showed 26 miRNAs upregulated in inoculated roots. Their predicted target genes are involved in the plant information processing system, which recognizes, percepts, and transmits signals, with higher representations in processes such as apoptosis and plant defense regulation. RNAseq data showed that predicted miRNA target genes were downregulated in tomato roots after 4, 7, 10, and 21 days post P. chlamydosporia inoculation. The differential expression of four miRNAs was further validated using qPCR analysis. The P. chlamydosporia endophytic lifestyle in tomato roots included an intricate network of miRNAs and targets. Data provide a first platform of DE tomato miRNAs after P. chlamydosporia colonization. They indicated that several miRNAs are involved in the host response to the fungus, playing important roles for its recognition as a symbiotic microorganism, allowing endophytism by modulating the host defense reaction. Data also indicated that endophytism affects tRNA fragmentation. This is the first study on miRNAs induced by P. chlamydosporia endophytism and related development regulation effects in Solanum lycopersicum.

Oxidative transformation of tunichromes - Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine.

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.

iTRAQ and RNA-Seq Analyses Provide New Insights into Regulation Mechanism of Symbiotic Germination of Dendrobium officinale Seeds (Orchidaceae).

Mycorrhizal fungi colonize orchid seeds and induce germination. This so-called symbiotic germination is a critical developmental process in the lifecycle of all orchid species. However, the molecular changes that occur during orchid seed symbiotic germination remain largely unknown. To better understand the molecular mechanism of orchid seed germination, we performed a comparative transcriptomic and proteomic analysis of the Chinese traditional medicinal orchid Dendrobium officinale to explore the change in protein expression at the different developmental stages during asymbiotic and symbiotic germination and identify the key proteins that regulate the symbiotic germination of orchid seeds. Among 2256 identified plant proteins, 308 were differentially expressed across three developmental stages during asymbiotic and symbiotic germination, and 229 were differentially expressed during symbiotic germination compared to asymbiotic development. Of these, 32 proteins were coup-regulated at both the proteomic and transcriptomic levels during symbiotic germination compared to asymbiotic germination. Our results suggest that symbiotic germination of D. officinale seeds shares a common signaling pathway with asymbiotic germination during the early germination stage. However, compared to asymbiotic germination, fungal colonization of orchid seeds appears to induce higher and earlier expression of some key proteins involved in lipid and carbohydrate metabolism and thus improves the efficiency of utilization of stored substances present in the embryo. This study provides new insight into the molecular basis of orchid seed germination.

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes.

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.

The Moro reaction: More than a reflex, a ritualized behavior of nonverbal communication.

OBJECTIVE: To propose a phylogenetic significance to the Moro reflex which remains unexplained since its publication in 1918 because both hands are free at the end of the gesture. METHOD: Among the 75 videos of healthy term newborns we have filmed in a research project on antenatal education to parenthood, we describe a sequence that clearly showed the successive movements of the Moro reflex and we report the occurrence of this reflex in the videos that were recorded from Time 0 of birth defined as the moment that lies between the birth of the thorax and the pelvis of the infant. RESULTS: The selected sequence showed the following succession of the newborn's actions: quick extension-adduction of both arms, the orientation of the body, head and eyes towards a human person, and full extension-abduction of both arms with spreading of the fingers, crying and a distressed face. There were 13 Moro reflexes between 2 and 14s from Time 0 of birth. We found a significant association between the occurrence of the Moro reflex and the placement of the newborn at birth in supine position on the mother's abdomen (p=0.002). DISCUSSION: The quick extension-adduction of both arms which started the sequence may be considered as a startle reflex controlled by the neural fear system and the arm extension-adduction which followed as a Moro reflex. The characteristics of all Moro reflexes were those of ritualization: amplitude, duration, stereotype of the gestures. This evolutionary process turns a physiological behavior, grasping in this case, to a non-verbal communicative behavior whose meaning is a request to be picked up in the arms. The gestures associated with the Moro reflex: crying and orientation of the body, head, and eyes towards a human person, are gestures of intention to communicate which support our hypothesis. The neural mechanism of the Moro reaction probably involves both the fear and the separation-distress systems. CONCLUSION: This paper proposes for the first time a phylogenetic significance to the Moro reflex: a ritualized behavior of nonverbal communication. Professionals should avoid stimulating the newborns' fear system by unnecessarily triggering Moro reflexes. Antenatal education should teach parents to respond to the Moro reflexes of their newborn infant by picking her up in their arms with mother talk.

Amygdalar neuronal activity mediates the cardiovascular responses evoked from the dorsolateral periaqueductal gray in conscious rats.

There is ample evidence that both lateral/dorsolateral periaqueductal gray (l/dlPAG) and basolateral amygdala (BLA) are essential for the regulation of the autonomic responses evoked during innate reactions to threatening stimuli. However, it is not well established to what extent the BLA regulates the upstream functional connection from the l/dlPAG. Here we evaluated the role of the BLA and its glutamatergic receptors in the cardiovascular responses induced by l/dlPAG stimulation in rats. We examined the influence of acute inhibition of the BLA, unilaterally, by injecting muscimol on the cardiovascular responses evoked by the injection of N-methyl D-aspartate (NMDA) into the l/dlPAG. We also evaluated the role of BLA ionotropic glutamate receptors in these responses by injecting antagonists of NMDA and AMPA/kainate receptor subtypes into the BLA. Our results show that the microinjection of NMDA in the BLA increased the mean arterial pressure (MAP) and heart rate (HR). Injection of NMDA into the l/dlPAG caused similar increases in these variables, which was prevented by the prior injection of muscimol, a GABAA agonist, into the BLA. Moreover, injection of glutamatergic antagonists (2-amino-5-phosphonopentanoate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) into the BLA reduced the increase in MAP and HR induced by l/dlPAG activation. Finally, the inhibition of the central amygdala neurons failed to reduce the cardiovascular changes induced by l/dlPAG activation. These results indicate that physiological responses elicited by l/dlPAG activation require the neuronal activity in the BLA. This ascending excitatory pathway from the l/dlPAG to the BLA might ensure the expression of the autonomic component of the defense reaction.

Failure of self-control: defense-like reactions during legume/rhizobia symbiosis.

Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. In this organ, symbiotic cells contain large numbers of bacteroids. Remarkably, this chronic infection does not trigger visible defense reactions. Despite the importance of this phenomenon for potential transfer of the symbiotic capacity to non-legume plants, the molecular mechanisms underlying this tolerance are not understood. We have characterized the dnf2 M. truncatula mutant blocked in the symbiotic process after bacterial infection of the symbiotic cells. Nodules formed by the mutant contain only few layers of infected cells. Furthermore, they exhibit defense-like reactions which clearly contrast with premature senescence frequently observed during inefficient symbioses. This atypical phenotype raises DNF2 as an exciting starting point to investigate the molecular basis of symbiotic repression of plant defenses.

Activation of Prophenoloxidase and Removal of Bacillus subtilis from the Hemolymph of Acheta domesticus (L. ) (Orthoptera: Gryllidae) / Ativação da Profenoloxidase e Remoção de Bacillus subtilis da Hemolinfa de Acheta domesticus (L. ) (Orthoptera: Gryllidae)

Fenoloxidase é considerada um importante mediador no sistema de defesa dos insetos contra patógenos e parasitóides. Essa enzima está presente na hemolinfa da maioria dos insetos estudados como uma proenzima inativa chamada profenoloxidase. Em Acheta domesticus (L.) a atividade da enzima foi estudada na presença de Bacillus subtilis. Injeções de bactéria na hemocele de ninfas desse inseto elevaram o nível de atividade da phenoloxidase. A taxa de aumento foi semelhante à observada nos insetos injetados com laminarin, um ativador da proenzima. A incubação de células de B. subtilis em soro com fenoloxidase ativada in vitro, e posterior injeção nos insetos, acelerou a remoção das bactérias da hemolinfa. A concentração das bactérias foi menor do que a observada nas ninfas injetadas apenas com bactéria em solução tampão. Por outro lado, ninfas injetadas com suspensão de bactéria incubada em soro com fenoloxidase inativada in vitro apresentaram alta concentração de bactéria na hemolinfa. Esses resultados indicam que B. subtilis é sensível à cascata de profenoloxidase de A. domesticus. Sugere-se que a fenoloxidase ativada adere à superfície das bactérias e aumenta a fixação dos hemócitos em torno das bactérias. Isso contribui para acelerar sua remoção da hemolinfa através da formação de nódulos.

Insect phenoloxidase is considered an important mediator in defense reaction against pathogens and parasites. It is present in the hemolymph of most insects as an inactive proenzyme, called prophenoloxidase. In Acheta domesticus (L.) the phenoloxidase activity was studied in the presence of Bacillus subtilis. The bacteria induced the activation of the prophenoloxidase and increased the level of phenoloxidase activity when injected into the hemocoel of A. domesticus nymphs. The level of increase was comparable to those observed in nymphs injected with laminarin, an activator of the proenzyme. Incubating B. subtilis in serum with phenoloxidase activated in vitro and then injecting into the insects accelerated the removal of the bacteria from the hemolymph in vivo. The concentration of bacteria was lower than those observed in nymphs injected with Mes buffer alone. In control insects, injection of soybean trypsin inhibitor lowered phenoloxidase activity and the removal of bacteria from the hemolymph. These results indicate that B. subtilis is sensitive to the prophenoloxidase cascade of A. domesticus. It is suggested that activated phenoloxidase could bind to the surface of bacteria and increase the adhesion of hemocytes to bacteria, and thus accelerating their removal by nodule formation.

Rhabditis pellio Schneider (nematoda) from the earthworm, Aporrectodea trapezoides Duges (Annelida).

Studies were conducted on the behavior of the nematode, Rhabditis pellio, in the earthworm, Aporrectodea trapezoides, from southern California. Juvenile and adult nematodes were found in the bladders and tubules of the metanephridia of the host. Similar nematodes that entered the coelom were encapsulated and incorporated into multiple capsules ("brown bodies"). It was demonstrated that this host response is an effective defense reaction since dead and dying nematodes, as well as living forms, were found in the capsules.