Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.     

Genome-Wide Identification of Gramineae Brassinosteroid-Related Genes and Their Roles in Plant Architecture and Salt Stress Adaptation

Brassinosteroid-related genes are involved in regulating plant growth and stress responses. However, systematic analysis is limited to Gramineae species, and their roles in plant architecture and salt stress remain unclear. In this study, we identified brassinosteroid-related genes in wheat, barley, maize, and sorghum and investigated their evolutionary relationships, conserved domains, transmembrane topologies, promoter sequences, syntenic relationships, and gene/protein structures. Gene and genome duplications led to considerable differences in gene numbers. Specific domains were revealed in several genes (i.e., HvSPY, HvSMOS1, and ZmLIC), indicating diverse functions. Protein-protein interactions suggested their synergistic functions. Their expression profiles were investigated in wheat and maize, which indicated involvement in adaptation to stress and regulation of plant architecture. Several candidate genes for plant architecture (ZmBZR1 and TaGSK1/2/3/4-3D) and salinity resistance (TaMADS22/47/55-4B, TaGRAS19-4B, and TaBRD1-2A.1) were identified. This study is the first to comprehensively investigate brassinosteroid-related plant architecture genes in four Gramineae species and should help elucidate the biological roles of brassinosteroid-related genes in crops.     

Polymorphisms in the Genes Coding for TLRs,NLRs and RLRs Are Associated with Clinical Parameters of Patients with Acute Myeloid Leukemia

Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) are major elements of the innate immune system that recognize pathogen-associated molecular patterns. Single-nucleotide polymorphisms (SNPs) in the TLR, NLR, and RLR genes may lead to an imbalance in the production of pro- and anti-inflammatory cytokines, changes in susceptibility to infections, the development of diseases, and carcinogenesis. Acute myeloid leukemia (AML) is a bone marrow malignancy characterized by uncontrolled proliferation of transformed myeloid precursors. We retrospectively analyzed 90 AML patients. We investigated the effect of fifteen SNPs located in the genes coding for RLR1 (rs9695310, rs10738889, rs10813831), NOD1 (rs2075820, rs6958571), NOD2 (rs2066845, rs2066847, rs2066844), TLR3 (rs5743305, rs3775296, 3775291), TLR4 (rs4986791, rs4986790), and TLR9 (rs187084, rs5743836). We observed that TLR4 rs4986791, TLR9 rs5743836, and NOD2 rs2066847 were associated with CRP levels, while RLR-1 rs10738889 was associated with LDH level. Furthermore, we found TLR3 rs5743305 AA to be more common in patients with infections. We also found TLR9 rs187084 C to be associated with more favorable risk, and RLR-1 rs9695310 GG with higher age at diagnosis. In conclusion, the current study showed that SNPs in the genes encoding TLRs, NLRs, and RLRs may be potential biomarkers in patients with AML.     

Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.)

The three-amino-acid-loop-extension (TALE) superfamily genes broadly existed in plants, which played important roles in plant growth, development and abiotic stress responses. In this study, we identified 68 Glycine max TALE (GmTALE) superfamily members. Phylogenetic analysis divided the GmTALE superfamily into the BEL1-like (BLH/BELL homeodomain) and the KNOX (KNOTTED-like homeodomain) subfamilies. Moreover, the KNOX subfamily could be further categorized into three clades (KNOX Class I, KNOX Class II and KNOX Class III). The GmTALE genes showed similarities in the gene structures in the same subfamily or clade, whose coding proteins exhibited analogous motif and conserved domain compositions. Besides, synteny analyses and evolutionary constraint evaluations of the TALE members among soybean and different species provided more clues for GmTALE superfamily evolution. The cis-element analyses in gene promoter regions and relevant gene expression profiling revealed different regulating roles of GmTALE genes during soybean plant development, saline and dehydration stresses. Genome-wide characterization, evolution, and expression profile analyses of GmTALE genes can pave the way for future gene functional research and facilitate their roles for applications in genetic improvement on soybean in saline and dehydration stresses.     

Rhamnolipid Biosurfactants as New Players in Animal and Plant Defense against Microbes

Rhamnolipids are known as very efficient biosurfactant molecules. They are used in a wide range of industrial applications including food, cosmetics, pharmaceutical formulations and bioremediation of pollutants. The present review provides an overview of the effect of rhamnolipids in animal and plant defense responses. We describe the current knowledge on the stimulation of plant and animal immunity by these molecules, as well as on their direct antimicrobial properties. Given their ecological acceptance owing to their low toxicity and biodegradability, rhamnolipids have the potential to be useful molecules in medicine and to be part of alternative strategies in order to reduce or replace pesticides in agriculture.     

The Innate Immune-Related Genes in Catfish

Catfish is one of the most important aquaculture species in America (as well as in Asia and Africa). In recent years, the production of catfish has suffered massive financial losses due to pathogen spread and breakouts. Innate immunity plays a crucial role in increasing resistance to pathogenic organisms and has generated increasing interest in the past few years. This review summarizes the current understanding of innate immune-related genes in catfish, including pattern recognition receptors, antimicrobial peptides, complements, lectins, cytokines, transferrin and gene expression profiling using microarrays and next generation sequencing technologies. This review will benefit the understanding of innate immune system in catfish and further efforts in studying the innate immune-related genes in fish.     

Genome-Wide Identification,Expansion, and Evolution Analysis of Homeobox Gene Family Reveals TALE Genes Important for Secondary Cell Wall Biosynthesis in Moso Bamboo (Phyllostachys edulis)

The TALE gene family is a subfamily of the homeobox gene family and has been implicated in regulating plant secondary growth. However, reports about the evolutionary history and function of the TALE gene family in bamboo are limited. Here, the homeobox gene families of moso bamboo Olyra latifolia and Bonia amplexicaulis were identified and compared. Many duplication events and obvious expansions were found in the TALE family of woody bamboo. PhTALEs were found to have high syntenies with TALE genes in rice. Through gene co-expression analysis and quantitative real-time PCR analysis, the candidate PhTALEs were thought to be involved in regulating secondary cell wall development of moso bamboo during the fast-growing stage. Among these candidate PhTALEs, orthologs of OsKNAT7, OSH15, and SH5 in moso bamboo may regulate xylan synthesis by regulating the expression of IRX-like genes. These results suggested that PhTALEs may participate in the secondary cell wall deposition in internodes during the fast-growing stage of moso bamboo. The expansion of the TALE gene family may be implicated in the increased lignification of woody bamboo when divergent from herbaceous bamboos.     

抗药性基因克隆技术研究进展

综述了近几年来抗药性基因克隆技术的研究进展。对RAPD-PCR、单链构象多态性(SSCP)、差异显示PCR(DDRT-PCR)、代表性差异分析技术(representational difference analysis，RDA)、抑制消减杂交法(suppression subtractive hybridization，SSH)、获得全长基因的消减杂交法(full length gene obtainable subtractive hybridization)、基因芯片技术、基因表达系统分析(serial analysis of gene expression，SAGE)的技术和从蛋白质水平克隆抗药性基因的双向电泳技术(two-dimensional gel electrophoresis)、噬菌体表面全套抗体库技术(phage display antibody repertoire library technique)、核糖体展示抗体库技术等方法的特点、优缺点及适用性进行了评述。     

A Putative Effector CcSp84 of Cytospora chrysosperma Localizes to the Plant Nucleus to Trigger Plant Immunity

Cytospora chrysosperma is the main causal agent of poplar canker disease in China, especially in some areas with poor site conditions. Pathogens secrete a large number of effectors to interfere the plant immunity and promote their infection and colonization. Nevertheless, the roles of effectors in C. chrysosperma remain poorly understood. In this study, we identified and functionally characterized a candidate effector CcSp84 from C. chrysosperma, which contained a nuclear localization signal motif at the C-terminal and was highly induced during infection stages. Transient expression of CcSp84 in Nicotiana benthamiana leaves could trigger cell death. Additionally, deletion of CcSp84 significantly reduced fungal virulence to the polar twigs, while no obvious defects were observed in fungal growth and sensitivity to H₂O₂. Confocal microscopy revealed that CcSp84 labeled with a green fluorescent protein (GFP) was mainly accumulated in the plant nucleus. Further analysis revealed that the plant nucleus localization of CcSp84 was necessary to trigger plant immune responses, including ROS accumulation, callose deposition, and induced expression of jasmonic acid and ethylene defense-related genes. Collectively, our results suggest that CcSp84 is a virulence-related effector, and plant nucleus localization is required for its functions.     

Ubiquitination of Receptorsomes,Frontline of Plant Immunity

Sessile plants are constantly exposed to myriads of unfavorable invading organisms with different lifestyles. To survive, plants have evolved plasma membrane-resident pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) to initiate sophisticated downstream immune responses. Ubiquitination serves as one of the most important and prevalent posttranslational modifications (PTMs) to fine-tune plant immune responses. Over the last decade, remarkable progress has been made in delineating the critical roles of ubiquitination in plant immunity. In this review, we highlight recent advances in the understanding of ubiquitination in the modulation of plant immunity, with a particular focus on ubiquitination in the regulation of receptorsomes, and discuss how ubiquitination and other PTMs act in concert to ensure rapid, proper, and robust immune responses.     

A TIR-NBS-LRR Gene MdTNL1 Regulates Resistance to Glomerella Leaf Spot in Apple

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is one of the most devastating apple diseases. Our previous study reported that the GLS resistance locus was defined on the chromosome 15 region. Here, we further found a single-nucleotide polymorphism (SNP) site (SNP_7309212) in the GLS resistance that was able to distinguish resistant cultivars (lines) from susceptible ones. On the basis of the SNP site, we cloned a TNL gene from the GLS resistant locus and named it MdTNL1 (NCBI Accession Number: {"type":"entrez-nucleotide","attrs":{"text":"ON402514","term_id":"2308172406","term_text":"ON402514"}}ON402514). This gene contains a toll/interleukin-1 receptor transmembrane domain (TIR), nucleotide-binding sites (NBS), and leucine-rich repeat (LRR) domain. Subcellular location indicated that MdTNL1 was expressed in the nucleus and cell membrane. Ectopic overexpression of MdTNL1 in Nicotiana benthamiana caused cell death. We further demonstrated allelic polymorphisms in MdTNL1. It is noteworthy that NBS and LRR domains of the MdTNL1 protein serve as the repository for generating allelic diversity. Quantitative real-time PCR (qRT-PCR) assay revealed that MdTNL1 was highly expressed in resistant apple cultivar ‘Fuji’ after inoculation with C. fructicola, whereas susceptible cultivar ‘Golden Delicious’ exhibited low expression after inoculation. Over-expression of MdTNL1-1 in susceptible apple fruits and leaves improved disease resistance, while in ‘Orin’ calli, silencing the MdTNL1-1 gene conversely decreased GLS resistance. In conclusion, we identified a GLS associated with SNP_7309212 and demonstrated that a TIR-NBS-LRR gene MdTNL1-1 positively regulates GLS resistance in apple.     

Advances in Biological Control and Resistance Genes of Brassicaceae Clubroot Disease-The Study Case of China

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae. It occurs in cruciferous crops exclusively, and causes serious damage to the economic value of cruciferous crops worldwide. Although different measures have been taken to prevent the spread of clubroot disease, the most fundamental and effective way is to explore and use disease-resistance genes to breed resistant varieties. However, the resistance level of plant hosts is influenced both by environment and pathogen race. In this work, we described clubroot disease in terms of discovery and current distribution, life cycle, and race identification systems; in particular, we summarized recent progress on clubroot control methods and breeding practices for resistant cultivars. With the knowledge of these identified resistance loci and R genes, we discussed feasible strategies for disease-resistance breeding in the future.     

Distinct Responses to Pathogenic and Symbionic Microorganisms: The Role of Plant Immunity

Plants must balance both beneficial (symbiotic) and pathogenic challenges from microorganisms, the former benefitting the plant and agriculture and the latter causing disease and economic harm. Plant innate immunity describes a highly conserved set of defense mechanisms that play pivotal roles in sensing immunogenic signals associated with both symbiotic and pathogenic microbes and subsequent downstream activation of signaling effector networks that protect the plant. An intriguing question is how the innate immune system distinguishes “friends” from “foes”. Here, we summarize recent advances in our understanding of the role and spectrum of innate immunity in recognizing and responding to different microbes. In addition, we also review some of the strategies used by microbes to manipulate plant signaling pathways and thus evade immunity, with emphasis on the use of effector proteins and micro-RNAs (miRNAs). Furthermore, we discuss potential questions that need addressing to advance the field of plant–microbe interactions.     

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer’s disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer’s disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.     

The Cutaneous Wound Innate Immunological Microenvironment

The skin represents the first line of defense and innate immune protection against pathogens. Skin normally provides a physical barrier to prevent infection by pathogens; however, wounds, microinjuries, and minor barrier impediments can present open avenues for invasion through the skin. Accordingly, wound repair and protection from invading pathogens are essential processes in successful skin barrier regeneration. To repair and protect wounds, skin promotes the development of a specific and complex immunological microenvironment within and surrounding the disrupted tissue. This immune microenvironment includes both innate and adaptive processes, including immune cell recruitment to the wound and secretion of extracellular factors that can act directly to promote wound closure and wound antimicrobial defense. Recent work has shown that this immune microenvironment also varies according to the specific context of the wound: the microbiome, neuroimmune signaling, environmental effects, and age play roles in altering the innate immune response to wounding. This review will focus on the role of these factors in shaping the cutaneous microenvironment and how this ultimately impacts the immune response to wounding.