Fine-tuning of the innate immune response by microRNAs

MicroRNAs (miRNAs) are emerging as potent regulators of many biological processes, including cellular differentiation and disease. Recently, miRNA has been directly involved in innate immunity and transduction signalling by Toll-like receptors and the ensuing cytokine response. In this review, we present an overview of what is currently known of the involvement of miRNA and RNA interference components in the fine-tuning of innate immune responses.     

Immunomodulation of jasmonate to manipulate the wound response

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.     

Influence of poly A-poly U on early events in the immune response in vitro

Adjuvant action of artificial homopolyribonucleotides and nucleic acid digests was studied in mouse spleen-cell cultures, stimulated with sheep red cells by the technique of Mishell and Dutton. Assays of antibody-forming cells were performed according to the Jerne and Nordin technique. Doublestranded poly A-poly U, and RNA digests had adjuvant activity over a wide dose range (0.01–100 μg/ml). DNA digests, single stranded polynucleotides (poly A, poly U, poly C) and mixtures of ribonucleosides and ribonucleotides had little or no enhancing effect at comparable doses. From our study of the kinetics of increase of plaque-forming cells in cultures treated with poly A-poly U, in conjunction with in vivo studies of others, we concluded that adjuvant action, during induction, indirectly increased the number of bone marrow-derived cells that responded to antigenic stimulation by increasing the effectiveness of the thymus-derived cells in the culture.     

Fine-tuning of the flavonoid and monolignol pathways during apple early fruit development

Planta - A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early...     

Increased sialylation and defucosylation of plasma proteins are early events in the acute phase response

Within hours of turpentine injection to stimulate the acute phase (AP) response in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of fucose decreases, each by about 60%. The two changes are inversely related (r = -0.97). The NeuAc/Gal ratio increases from the normal 0.75 to 1.0 on day 2 of the AP. Whereas 50% of the isolated oligosaccharides of normal plasma proteins are retarded on immobilized Ricinus communis agglutinin, those from day 2 AP plasma fail to do so. This indicates that NeuAc caps the normally Gal-terminated chains. alpha1-Acid glycoprotein (a positive AP protein), alpha1-macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also show similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2-Macroglobulin, which arises only during the AP, does not contain significant amounts of Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of plasma proteins, and binding is increased during the AP response. Maackia amurensis lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding decreases during the AP response. These results show that: (1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3) the oligosaccharides of even the non-AP and negative AP proteins are modified. These changes are distinct from the elevation in the levels of protein-bound monosaccharides and the altered concanavalin A-binding profile the oligosaccharides of AP proteins acquire in diseases.     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

Calcium changes and the response to methyl jasmonate in rice lodicules during anthesis

Summary. Potassium pyroantimonate precipitation was used to locate loosely bound calcium in rice (Oryza sativa L.) lodicules before and after anthesis, and flowering of panicles was accelerated by treatment with methyl jasmonate. From 1 day to 4 h before anthesis, the number of calcium precipitates in the cell walls and vacuole membranes decreased gradually, whereas they increased remarkably in the cytoplasm and nucleolus. At the beginning of anthesis, the number of calcium granules in lodicules reduced sharply, but there was a large accumulation of flocculent precipitates in the vacuoles. After anthesis, the flocculent precipitates decreased in number until they disappeared, whereas the granular precipitates started to accumulate once again. The rice florets treated with 2 mM methyl jasmonate were induced to open within 10–30 min and they then closed 0.5–1 h later. The nucleolus, cytoplasm, and vacuole membrane of the lodicule cells contained many calcium granules during flowering, although the cell walls lacked calcium. At 1 h after treatment, the number of calcium granules had decreased, while flocculent precipitates were regularly observed in the nondegenerated cells. At 6 h after treatment, calcium grains started to reappear in the cell walls. These changes in calcium precipitates before and after anthesis indicate that the opening and closing of florets correlates with the calcium level in lodicule cells. In addition, excised panicles, with florets judged to be nearing anthesis, were soaked in 2–200 mM EGTA solution for 2 min after treatment with 2 mM methyl jasmonate. The results indicate that EGTA had an antagonistic effect on the methyl jasmonate-induced floret opening in rice. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Regulation of early events in chromosome replication

Eukaryotic genomes are replicated from large numbers of replication origins distributed on multiple chromosomes. The activity of these origins must be coordinated so that the entire genome is efficiently and accurately replicated yet no region of the genome is ever replicated more than once. The past decade has seen significant advances in understanding how the initiation of DNA replication is regulated by key cell-cycle regulators, including the cyclin dependent kinases (CDKs) and the anaphase promoting complex/cyclosome (APC/C). The assembly of essential prereplicative complexes (pre-RCs) at origins only occurs when CDK activity is low and APC/C activity is high. Origin firing, however, can only occur when the APC/C is inactivated and CDKs become active. This two step mechanism ensures that no origin can fire more than once in a cell cycle. In all eukaryotes tested, CDKs can contribute to the inhibition of pre-RC assembly. This inhibition is characterised both by high degrees of redundancy and evolutionary plasticity. Geminin plays a crucial role in inhibiting licensing in metazoans and, like cyclins, is inactivated by the APC/C. Strategies involved in preventing re-replication in different organisms will be discussed.     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

Analyses of phosphorylation events in the rubella virus capsid protein: role in early replication events

The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.     

Immunosuppressive activity of antibody directed against endogenous C-type virus interferes with early events of the immune response.

We have analyzed the effects of an antiserum prepared against BALB/c endogenous xenotropic C-type virus on the humoral immune response of mice. Both in vivo and in vitro, this serum suppresses the response to sheep red blood cells, an effect that can be absorbed out by purified BALB/c xenotropic C-type virus or Friend leukemia virus, but not by Rous sarcoma virus. The serum produces its maximum effect when administered together with or before the antigen, but not 24 hr later. This suggests that it acts on an early event of the immune response. Evidence is presented to show that the critical viral antigen is expressed before the spleen cells are experimentally stimulated by antigen. The same immunosuppressive effect was observed in a variety of mouse strains, including the high-leukemia incidence AKR strain and virus-free 129/J mice, indicating that it is independent of the expression of endogenous virus. The finding that a viral antigen is involved in the transition from a resting to a dividing lymphocyte is discussed with respect to viral involvement in leukemia.