Alterations of iron distribution in Arabidopsis tissues infected by Dickeya dadantii

Dickeya dadantii is a plant‐pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana. Iron distribution in infected A. thaliana was investigated at the cellular scale using the Perls'–diaminobenzidine–H₂O₂ (PDH) method. Iron visualization during infection reveals a loss of iron from cellular compartments and plant cell walls. During symptom progression, two distinct zones are clearly visible: a macerated zone displaying weak iron content and a healthy zone displaying strong iron content. Immunolabelling of cell wall methylated pectin shows that pectin degradation is correlated with iron release from cell walls, indicating a strong relationship between cell wall integrity and iron in plant tissues. Using a D. dadantii lipopolysaccharide antibody, we show that bacteria are restricted to the infected tissue, and that they accumulate iron in planta. In conclusion, weak iron content is strictly correlated with bacterial cell localization in the infected tissues, indicating a crucial role of this element during the interaction. This is the first report of iron localization at the cellular level during a plant–microbe interaction and shows that PDH is a method of choice in this type of investigation.     

Role of Dickeya dadantii 3937 chemoreceptors in the entry to Arabidopsis leaves through wounds

Chemotaxis enables bacteria to move towards an optimal environment in response to chemical signals. In the case of plant‐pathogenic bacteria, chemotaxis allows pathogens to explore the plant surface for potential entry sites with the ultimate aim to prosper inside plant tissues and to cause disease. Chemoreceptors, which constitute the sensory core of the chemotaxis system, are usually transmembrane proteins which change their conformation when sensing chemicals in the periplasm and transduce the signal through a kinase pathway to the flagellar motor. In the particular case of the soft‐rot pathogen Dickeya dadantii 3937, jasmonic acid released in a plant wound has been found to be a strong chemoattractant which drives pathogen entry into the plant apoplast. In order to identify candidate chemoreceptors sensing wound‐derived plant compounds, we carried out a bioinformatics search of candidate chemoreceptors in the genome of Dickeya dadantii 3937. The study of the chemotactic response to several compounds and the analysis of the entry process to Arabidopsis leaves of 10 selected mutants in chemoreceptors allowed us to determine the implications of at least two of them (ABF‐0020167 and ABF‐0046680) in the chemotaxis‐driven entry process through plant wounds. Our data suggest that ABF‐0020167 and ABF‐0046680 may be candidate receptors of jasmonic acid and xylose, respectively.     

Immunological memory within the innate immune system

Immune memory has traditionally been the domain of the adaptive immune system, present only in antigen‐specific T and B cells. The purpose of this review is to summarize the evidence for immunological memory in lower organisms (which are not thought to possess adaptive immunity) and within specific cell subsets of the innate immune system. A special focus will be given to recent findings in both mouse and humans for specificity and memory in natural killer (NK) cells, which have resided under the umbrella of innate immunity for decades. The surprising longevity and enhanced responses of previously primed NK cells will be discussed in the context of several immunization settings.     

Beta-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana

Most plant glycoproteins contain substantial amounts of paucimannosidic N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. We now demonstrate that two β-N-acetylhexosaminidases (HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. Total N-glycan analysis of hexo knock-out plants revealed that HEXO1 and HEXO3 contribute equally to the production of paucimannosidic N-glycans in roots, whereas N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Because hexo1 hexo3 double mutants do not display any obvious phenotype even upon exposure to different forms of abiotic or biotic stress, it should be feasible to improve the quality of glycoprotein therapeutics produced in plants by down-regulation of endogenous β-N-acetylhexosaminidase activities.     

Commonly invasive serotypes of Streptococcus pneumoniae trigger a reduced innate immune response compared with serotypes rarely responsible for invasive infection

Although there are more than 90 serotypes of Streptococcus pneumoniae (or pneumococcus), it is not understood why a small number of serotypes account for most invasive infections. To investigate the human innate immune response triggered by different pneumococcal serotypes, monocyte-derived macrophages were exposed to a group of commonly and rarely invasive pneumococcal clinical isolates and tumor necrosis factor (TNF)-alpha production was measured. Commonly invasive pneumococcal serotypes triggered significantly less TNF-alpha production than serotypes rarely responsible for invasive infection (P<0.004). These data indicate that one factor influencing the invasive potential of a pneumococcal serotype is the magnitude of innate immune-mediated TNF-alpha production triggered by exposure to the organism and suggest that the integrated host response generated against commonly invasive pneumococcal serotypes may be less effective than the response directed against rarely invasive serotypes.     

The interaction between maternal stress and the ontogeny of the innate immune system during teleost embryogenesis: implications for aquaculture practice

The barrier defences and acellular innate immune proteins play critical roles during the early‐stage fish embryos prior to the development of functional organ systems. The innate immune proteins in the yolk of embryos are of maternal origin. Maternal stress affects the maternal‐to‐embryo transfer of these proteins and, therefore, environmental stressors may change the course of embryo development, including embryonic immunocompetency, via their deleterious effect on maternal physiology. This review focuses on the associations that exist between maternal stress, maternal endocrine disturbance and the responses of the acellular innate immune proteins of early‐stage fish embryos. Early‐stage teleostean embryos are dependent upon the adult female for the formation of the zona pellucida as an essential barrier defence, for their supply of nutrients, and for the innate immunity proteins and antibodies that are transferred from the maternal circulation to the oocytes; maternally derived hormones are also transferred, some of which (such as cortisol) are known to exert a suppressive action on some aspects of the immune defences. This review summarizes what is known about the effects of oocyte cortisol content on the immune system components in early embryos. The review also examines recent evidence that embryonic cells during early cleavage have the capacity to respond to increased maternal cortisol transfer; this emphasizes the importance of maternal and early immune competence on the later life of fishes, both in the wild and in intensive culture.     

Plasma lipoproteins are important components of the immune system

Plasma lipoproteins (VLDL, LDL, Lp[a] and HDL) function primarily in lipid transport among tissues and organs. However, cumulative evidence suggests that lipoproteins may also prevent bacterial, viral and parasitic infections and are therefore a component of innate immunity. Lipoproteins can also detoxify lipopolysaccharide and lipoteichoic acid. Infections can induce oxidation of LDL, and oxLDL in turn plays important anti‐infective roles and protects against endotoxin‐induced tissue damage. There is also evidence that apo(a) is protective against pathogens. Taken together, the evidence suggests that it might be valuable to introduce the concept that plasma lipoproteins belong in the realm of host immune response.     

The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c‐di‐GMP network

Bacteria use signal transduction systems to sense and respond to their external environment. The two‐component system CpxA/CpxR senses misfolded envelope protein stress and responds by up‐regulating envelope protein factors and down‐regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS‐inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c‐di‐GMP regulators were also up‐regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c‐di‐GMP phenotypes in biofilm formation and swimming. Increased production of cellular c‐di‐GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c‐di‐GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.     

Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. Here, we show that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. Our results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Antimicrobial peptides: key components of the innate immune system

Life-threatening infectious diseases are on their way to cause a worldwide crisis, as treating them effectively is becoming increasingly difficult due to the emergence of antibiotic resistant strains. Antimicrobial peptides (AMPs) form an ancient type of innate immunity found universally in all living organisms, providing a principal first-line of defense against the invading pathogens. The unique diverse function and architecture of AMPs has attracted considerable attention by scientists, both in terms of understanding the basic biology of the innate immune system, and as a tool in the design of molecular templates for new anti-infective drugs. AMPs are gene-encoded short (<100 amino acids), amphipathic molecules with hydrophobic and cationic amino acids arranged spatially, which exhibit broad spectrum antimicrobial activity. AMPs have been the subject of natural evolution, as have the microbes, for hundreds of millions of years. Despite this long history of co-evolution, AMPs have not lost their ability to kill or inhibit the microbes totally, nor have the microbes learnt to avoid the lethal punch of AMPs. AMPs therefore have potential to provide an important breakthrough and form the basis for a new class of antibiotics. In this review, we would like to give an overview of cationic antimicrobial peptides, origin, structure, functions, and mode of action of AMPs, which are highly expressed and found in humans, as well as a brief discussion about widely abundant, well characterized AMPs in mammals, in addition to pharmaceutical aspects and the additional functions of AMPs.     

Interaction between nanomaterials and the innate immune system across evolution

Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte- and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function

Chronic inflammation in older individuals is thought to contribute to inflammatory, age‐related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross‐sectional study involving 91 healthy male (aged 20–84 years, median 52.4) and 55 female (aged 20–82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age‐related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age‐related diseases.     

拟南芥SSR检测体系的优化

以拟南芥(Arabidopsis thaliana)幼苗为实验材料,采用单因素筛选法及L16(45)正交实验方法,对简单序列重复(SSR)技术中聚合酶链式反应(PCR)组分、扩增程序、电泳检测等环节进行优化。优化25μl反应体系为:1×PCR Buffer、20ng模板DNA、1.5mmol.L-1Mg2+、0.3μmol·L-1引物、150μmol·L-1dNTPs和1.0U Taq DNA聚合酶。扩增程序为:94℃预变性5min,94℃变性30s,57℃退火30s,72℃延伸45s,共30个循环,72℃延伸10min。用非变性聚丙烯酰胺凝胶(EB染色)电泳检测并取得较好效果。利用该体系进行扩增,所得谱带清晰、稳定、非特异性带少。