Genome-wide identification and characterization of putative effectors in Heterodera schachtii that suppress plant immune response

The sugar beet cyst nematode(Heterodera schachtii) is one of the most destructive pathogens in sugar beet production, which causes serious economic losses every year. Few molecular details of effectors of H. schachtii parasitism are known. We analyzed the genome and transcriptome data of H. schachtii and identified multiple potential predicted proteins. After filtering out predicted proteins with high homology to other plant-parasitic nematodes, we performed functional validation of the remaining effector proteins. 37 putative effectors of H. schachtii were screened based on the Nicotiana benthamiana system for identifying the effectors that inhibit plant immune response, eventually determines 13 candidate effectors could inhibit cell death caused by Bax. Among the 13 effectors, nine have the ability to inhibit GPA2/RBP1-induced cell death. All 13 effectortriggered immunity(ETI) suppressor genes were analyzed by qRT-PCR and confirmed to result in a significant downregulation of one or more defense genes during infection compared to empty vector. For in situ hybridization,13 effectors were specifically expressed and located in esophageal gland cells. These data and functional analysis set the stage for further studies on the interaction of H. schachtii with host and H. schachtii parasitic control.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops.Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear.A glycine-rich nuclear effector,VdCE51,was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum.This effector was a required factor for full virulence of V.dahliae,and its nuclear localization was a requisite for suppressing plant immunity.The thioredoxin GhTRXH2,identified as a positive regulator of plant immunity,was a host target of VdCE51.Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes,and the SA signaling pathway by inhibiting the accumulation of GhTRXH2,thus suppressing plant immunity.

A novel chorismate mutase effector secreted from root-knot nematode Meloidogyne enterolobii manipulates plant immunity to promote parasitism

Meloidogyne spp.is an economically important plant-parasitic nematode distributed worldwide.To fight with host immune system for successful parasitism,plant parasitic nematodes secrete effectors to promote infection.In this study,we identified one chorismate mutase(CM)effector from M.enterolobii,named Me-CM.Spatial and temporal expression assays exhibited Me-cm is expressed in esophageal glands and up-regulated at parasitic-stage juveniles.Me-CM affects the pathogenicity of M.enterolobii based on the reduced infection rate,number of galls,egg masses,eggs per mass and multiplication rate collected from RNA silencing experiments.We showed that Me-CM localized in the cytoplasm and nucleus of plant cells and decreased the expression level of the marker gene PR1 of salicylic acid(SA)pathway.Besides,constitutive expression of Me-cm in Arabidopsis thaliana significantly reduced salicylic acid concentration.These results suggested that M.enterolobii may secrete effector Me-CM to fight with plantimmunesystemsvia regulating SA signaling pathway when interacting with host plants,ultimately facilitating parasitism.

The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

核盘菌致病机制研究进展

核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)是世界性的死体营养型植物病原真菌,寄主范围极广,所引起的大豆和油菜等菌核病会导致农业生产的巨大经济损失.核盘菌致病机制复杂,不仅具有直接杀死细胞的典型死体营养阶段,也包含需抑制植物免疫的短暂活体营养期.核盘菌具有种类繁多的致病因子,包括介导侵染结构形成或抗逆能力的关键调控因子、降解植物细胞组分的水解酶、草酸、诱导植物细胞死亡或抑制植物免疫的效应蛋白等.综述核盘菌侵染模型,总结各种致病因子,尤其是效应蛋白在核盘菌致病中的作用,并结合最新研究结果对核盘菌致病新机制进行展望,为作物菌核病的防控提供理论依据.

圣女果串采收末端执行器的设计与实验

为提高圣女果采摘效率和降低果实损伤,设计了圣女果串采收末端执行器。其末端结构采用平面四连杆驱动夹持剪切机构,夹持机构采用自补偿设计,使夹持与剪切动作一体化完成。同时,基于螺旋理论,建立接触模型,验证分析了夹持结构的力封闭性,并对整体装置进行了动力学和运动学分析与仿真。室内实验结果表明:整串采摘的夹持成功率为100%,剪切成功率为86.7%。

水蜜桃套袋机器人及其末端执行器研制

农业智能机器人的发展为传统农业带来了新的发展机遇。特别是在季节性劳动力需求大的农业生产中,迫切需要智能机械取代人工劳动。在研究水蜜桃幼果套袋过程中,提出了一种具有创新性的水蜜桃幼果套袋机器人,并根据人工套袋的方式,开发了一款套袋专用的末端执行器。该末端执行器的关键部件包括取送袋部件、套袋开口部件和封口部件。通过对末端执行器控制流程的分析,完成一个水蜜桃幼果的套袋任务需要15.4 s。

小麦矮腥黑粉菌g6843蛋白生物信息学分析、毒性验证及亚细胞定位

引起小麦矮腥黑穗病的病原菌小麦矮腥黑粉菌(Tilletia controversa Kühn)是一类严重危害麦类作物的真菌。为解析小麦矮腥黑粉菌与植物互作机制,本研究基于前期全基因组测序结果,筛选出候选效应蛋白g6843,并运用生物信息学分析预测其生物学功能。通过农杆菌介导的烟草瞬时表达系统和激光共聚焦显微镜技术,对g6843蛋白的毒性和亚细胞定位进行功能验证。生物信息学预测结果表明,g6843基因全长为1413 bp,共编码470个氨基酸,分子量为49626.47 Da,氨基酸等电点为5.58;该蛋白是亲水性蛋白,包含23个信号肽,不存在跨膜结构,在第33~第223个氨基酸之间和第268~第466个氨基酸之间含有α/β水解酶家族蛋白功能域,属于稳定的偏酸性蛋白;其中二级结构的主要构成原件为无规卷曲,其次是α螺旋,三级结构复杂。毒性验证观察到g6843蛋白不能抑制由Bcl-2 associated X protein(BAX)凋亡蛋白引起的烟草叶片坏死。激光共聚焦显微镜观察结果显示g6843蛋白定位于细胞膜和细胞核。本研究结果为进一步研究g6843蛋白在植物与病原体相互作用中的功能提供了重要的理论依据。

基于力反馈设备的工业机器人遥操作装配系统实验研究

采用工业机器人执行某些危险或复杂环境下的装配任务时,需要通过遥操作机器人进行装配,为此,提出了一种基于力反馈设备的工业机器人遥操作装配系统。首先,基于该装配系统,建立了遥操作力反馈设备位姿坐标系与机器人末端坐标系间的位姿转换矩阵,将主端的位姿信息映射到机器人末端执行器,从而驱动机器人完成了装配任务;然后,引入了力位混合控制器,并结合力和位置控制策略,通过选择六维方向控制模式,实现了对机器人位姿进行柔顺控制的目的;最后,搭建了机器人遥操作装配平台,进行了工业机器人的装配实验,对该装配系统能否满足工业机器人遥操作装配的需求进行了验证。研究结果表明:采用该工业机器人遥操作装配的系统中,平均位置误差为1.121 mm,平均角度误差为0.589°,具有良好的控制精度;采用该力位混合控制器后,装配的成功率提升了30%,并且成功装配的平均时间缩短了6.7 s,有效提升了装配的完成效率和完成质量。

Yersinia type Ⅲ effectors perturb host innate immune responses

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type Ⅲ secretion system(T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp.(Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gramnegative bacteria that share in common a 70 kb virulence plasmid which encodes the T3 SS. Translocation of the Yersinia effector proteins(YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.

Design of Drilling and Riveting Multi-functional End Effector for CFRP and Aluminum Components in Robotic Aircraft Assembly

To fulfill the demands for higher quality,efficiency and flexibility in aviation industry,a multi-functional end effector is designed to automate the drilling and riveting processes in assembling carbon fiber reinforced polymer(CFRP)and aluminum components for a robotic aircraft assembly system.To meet the specific functional requirements for blind rivet installation on CFRP and aluminum materials,additional modules are incorporated on the end effector aside of the basic processing modules for drilling.And all of these processing modules allow for a onestep-drilling-countersinking process,hole inspection,automatic rivet feed,rivet geometry check,sealant application,rivet insertion and installation.Besides,to guarantee the better quality of the hole drilled and joints riveted,several online detection and adjustment measures are applied to this end effector,including the reference detection and perpendicular calibration,which could effectively ensure the positioning precision and perpendicular accuracy as demanded.Finally,the test result shows that this end effector is capable of producing each hole to a positioning precision within ±0.5 mm,aperpendicular accuracy within 0.3°,a diameter tolerance of H8,and a countersink depth tolerance of±0.01 mm.Moreover,it could drill and rivet up to three joints per minute,with acceptable shearing and tensile strength.

IN VITRO AND IN VIVO CHEMOTACTIC EFFECT OF MIP-1a GENE TRANSFECTED TUMOR VACCINE ON IMMUNE EFFECTOR CELLS

Vaccination with chemokine genetransfected tumor cells may be a new apporach to cancer treatment. Macrohage inflammatory protein1α (MIP1α) is a new type of chemokines which has chemotactic activity on effector cells. In the present study, the B16 melanoma cells were transfected with recombinant adenovirus harboring murine MIP1α gene. The biological characteristics of the MIP1α gene transfected B16 melanoma cells were investigated. The level of MIP1α in the supernatant of genetransfected melanoma cells was 368±24 ng/ml/106/24hr. This supernatant showed strong chematactic activity for NK cells, CD4+ T cells, CD8+ T cells or the freshly isolated peritoneal macrophages. Though the in vitro growth were not altered, the tumorigenicity of the genetransfected B16 melanoma cells decreased signicantly. The infiltration of inflammatory cells into the tumor mass formed by MIP1α genetransfected B16 cells were much more obvious than that by wildtype B16 cells or B16 cells transfected with the control gene. However, the survival time of the mice bearing B16 melanoma cells transfected with MIP1α gene was not prolonged and the NK, CTL activity remianed unchanged. We suggested that the in vivo phenomenon may be related to the high toxicity of the MIP1 α as a strong nonspecific inflammatory mediator.

Puccinia triticina effector protein Pt_21 interacts with wheat thaumatin-like protein TaTLP1 to inhibit its antifungal activity and suppress wheat apoplast immunity

Puccinia triticina(Pt), as the causal agent of wheat leaf rust, employs a plethora of effector proteins to modulate wheat immunity for successful colonization. Understanding the molecular mechanisms underlying Pt effector-mediated wheat susceptibility remains largely unexplored. In this study, an effector Pt_21 was identified to interact with the apoplast-localized wheat thaumatin-like protein TaTLP1 using a yeast two-hybrid assay and the Pt_21-TaTLP1 interaction was characterized. The interaction between Pt_21 and TaTLP1 was validated by in vivo co-immunoprecipitation assay. A TaTLP1 variant,TaTLP1C71A, that was identified by the site-directed mutagenesis failed to interact with Pt_21. Pt_21was able to suppress Bax-mediated cell death in leaves of Nicotiana benthamiana and inhibit TaTLP1-mediated antifungal activity. Furthermore, infiltration of recombinant protein Pt_21 into leaves of transgenic wheat line overexpressing TaTLP1 enhanced the disease development of leaf rust compared to that in wild-type leaves. These findings demonstrate that Pt_21 suppresses host defense response by directly targeting wheat TaTLP1 and inhibiting its antifungal activity, which broadens our understanding of the molecular mechanisms underlying Pt effector-mediated susceptibility in wheat.

A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Neural regeneration after peripheral nerve injury repair is a system remodelling process of interaction between nerves and terminal effector

In China, there are approximately 20 million people suffering from peripheral nerve injury and this number is increasing at a rate of 2 million per year. These patients cannot live or work independently and are a heavy responsibility on both family and society because of extreme disability and dysfunction caused by peripheral nerve injury （PNI）. Thus, repair of PNI has become a major public health issue in China.

Design Schemes and Comparison Research of the End-effector of Large Space Manipulator

The end-effector of the large space manipulator is employed to assist the manipulator in handling and manipulating large payloads on orbit.Currently,there are few researches about the end-effector,and the existing end-effectors have some disadvantages,such as poor misalignment tolerance capability and complex mechanical components.According to the end positioning errors and the residual vibration characters of the large space manipulators,two basic performance requirements of the end-effector which include the capabilities of misalignment tolerance and soft capture are proposed.And the end-effector should accommodate the following misalignments of the mechanical interface.The translation misalignments in axial and radial directions and the angular misalignments in roll,pitch and yaw are ±100 mm,100 mm,±10°,±15°,±15°,respectively.Seven end-effector schemes are presented and the capabilities of misalignment tolerance and soft capture are analyzed elementarily.The three fingers-three petals end-effector and the steel cable-snared end-effector are the most feasible schemes among the seven schemes,and they are designed in detail.The capabilities of misalignment tolerance and soft capture are validated and evaluated,through the experiment on the micro-gravity simulating device and the dynamic analysis in ADAMS software.The results show that the misalignment tolerance capabilities of these two schemes could satisfy the requirement.And the translation misalignment tolerances in axial and radial directions and the angular misalignment tolerances in roll,pitch and yaw of the steel cable-snared end-effector are 30mm,15mm,6°,3° and 3° larger than those of the three fingers-three petals end-effector,respectively.And the contact force of the steel cable-snared end-effector is smaller and smoother than that of the three fingers-three petals end-effector.The end-effector schemes and research methods are beneficial to the developments of the large space manipulator end-effctor and the space docking mechanism.

Regulation of neuroinflammatory properties of glial cells by T cell effector molecules

Multiple sclerosis （MS） is a chronic inflammatory and neurodegenerative disorder that is thought to be mediated by autoreactive T lymphocytes that find their way into the central nervous system （CNS）. The pathological mechanism of MS is still being elucidated but it involves complex interactions between infiltrating immune cells and resi- dent glial cells within the CNS that culminate into strong neuroinflammation and axonal damage.

CD_4^(+) EFFECTOR CELLS DEFAULT TO THE TH2 PATHWAY IN INTERFERONγ-DEFICIENT MICE INFECTED WITH LEISHMANIA MAJOR

Mice with homologous disruption of the interferon γ(IFN-γ) gene on the C57BL/6 background were infected witly Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice,deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6￣8 wk.Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-γ and lymphotoxin,tipical of T helper type 1(Th1) cells, the knockout mice developed CD+4 cells that contained transcripts for interleukin 4(IL-4),IL-5,and IL13,typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-γ or IL-4 production by T cells in similar frequencies in the respective groups of mice,and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-γ.

Bioinformatic Analysis of Potential Pathogenicity Effectors of <i>Candidatus</i>Liberibacter asiaticus, Causal Agent of Citrus Huanglongbing

Huanglongbing (HLB) or citrus greening is currently the most important citrus disease, caused by the bacterium Candidatus Liberibacter asiaticus (CLas). The impossibility of isolating it causes understanding its pathogenic mechanisms to be a complicated task. Recent studies identified 16 proteins with the signal peptide needed to be secreted in the plant and cause the disease. The present study aims to perform a bioinformatic analysis of these proteins with the function prediction approach by gene ontology (GO) and the detection of conserved domains. It was observed that of the 16 proteins analyzed not all are found in different infective strains reported in the literature. The GO analysis allowed us to relate different proteins with the biological process of energy and pathogenic activity, especially CLIBASIA_03315 and CLIBASIA_05115, respectively. The domain analysis allowed the observation of a β-CA domain, tentatively related to the damage caused to the chloroplast and a PAAR domain associated with the T6SS secretory system. Our results provide information on the possible function of potential pathogenicity effectors in CLas.

Memory AND effector cells in children with bacterial infections of the gastrointestinal and respiratory tracts

Infections in infants and children under five years of age are a public health in México and are one of the major causes of death. Methods In this study, lymphocyte immunophenotyping for CD3+ (T-cells), CD3+CD4+, CD3+CD8+, CD3+CD19+, CD3+CD16/56+, CD45RA+, CD45RO+, CD62L- and CD28- were determined in the whole blood of gastrointestinal and respiratory bacterial infected children, using a four-color flow cytometry technique. Results: Our data showed that the percentages and the absolute numbers of monocytes and granulocytes are increased in infected children, when compared to the control group. Similarly, we observed increases in the percentages of B lymphocytes, CD8+ cells, memory T cells (CD4+CD45RO+ and CD8+CD45RO+) and effector lymphocytes (CD4+CD62L? and CD8+CD28?) in infected children compared with the control group. In contrast, naive T cells were decreased in the bacterial infected children relative to the control group. Additionally, we used ELISA assays to identify the pathogen agent in gastrointestinal and respiratory infection. Comparing different types of infection, we found that the children with respiratory bacterial infections had higher percentages of B lymphocytes, and cytotoxic lymphocytes (CD8+CD28-);and the children with gastrointestinal infections had higher percentages of CD3+ lymphocytes and effector cells (CD4+CD62L-). Conclusions The increase in B lymphocytes and CD8+CD28- cells in the children with respiratory infections and the increase of T lymphocytes and CD4+CD62L- cells in the children with gastrointestinal bacterial infections indicate that both cellular and humoral responses coincide, and both responses are necessary for eliminating the pathogen.