A plant effector‐triggered immunity signaling sector is inhibited by pattern‐triggered immunity

Since signaling machineries for two modes of plant‐induced immunity, pattern‐triggered immunity (PTI) and effector‐triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta. These observations led us to discovery of a network‐buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI‐Mediating and PTI‐Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.     

两类免疫受体强强联手筑牢植物免疫防线

植物先天免疫系统在抵御病原菌入侵过程中发挥至关重要的作用, 主要包括两个层次, 即病原菌相关分子模式和效应因子分别触发的PTI和ETI免疫反应。PTI和ETI分别由植物细胞膜表面模式识别受体(PRRs)和胞内免疫受体(NLRs)激活, 具有特异的激活机制, 但是两者激活的下游免疫事件相互重叠。PTI和ETI是否为泾渭分明的两道防线, 以及ETI与PTI下游事件为何如此相似, 一直是植物免疫领域最受关注的问题之一。最近, 中国科学院分子植物科学卓越创新中心辛秀芳团队与合作者利用拟南芥(Arabidopsis thaliana)与丁香假单胞杆菌(Pseudomonas syringae)互作系统对PTI和ETI在机制上的联系进行了研究。他们发现PRRs和共受体参与ETI, 而活性氧的产生是联系PRRs和NLRs所介导的免疫早期信号事件。他们还发现NLRs信号能够迅速增强PTI关键因子的转录和蛋白水平, PTI的增强在ETI免疫反应中不可或缺。该研究从机制上解析了植物免疫领域中长期悬而未决的PTI与ETI相似性之谜, 是该领域的一项突破性进展, 为未来作物分子设计育种提供了新的启示。     

Toward understanding of rice innate immunity against Magnaporthe oryzae

The blast fungus, Magnaporthe oryzae, causes serious disease on a wide variety of grasses including rice, wheat and barley. The recognition of pathogens is an amazing ability of plants including strategies for displacing virulence effectors through the adaption of both conserved and variable pathogen elicitors. The pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were reported as two main innate immune responses in plants, where PTI gives basal resistance and ETI confers durable resistance. The PTI consists of extracellular surface receptors that are able to recognize PAMPs. PAMPs detect microbial features such as fungal chitin that complete a vital function during the organism’s life. In contrast, ETI is mediated by intracellular receptor molecules containing nucleotide-binding (NB) and leucine rich repeat (LRR) domains that specifically recognize effector proteins produced by the pathogen. To enhance crop resistance, understanding the host resistance mechanisms against pathogen infection strategies and having a deeper knowledge of innate immunity system are essential. This review summarizes the recent advances on the molecular mechanism of innate immunity systems of rice against M. oryzae. The discussion will be centered on the latest success reported in plant–pathogen interactions and integrated defense responses in rice.     

The Pseudomonas syringae type III effector HopG1 targets mitochondria,alters plant development and suppresses plant innate immunity

The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector‐triggered immunity (ETI) and pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N‐terminal two‐thirds was sufficient for mitochondrial localization. A HopG1–GFP fusion lacking HopG1's N‐terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N‐terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1's target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions.     

Pseudomonas syringae pv. phaseolicola effector HopF1 inhibits pathogen-associated molecular pattern-triggered immunity in a RIN4-independent manner in common bean (Phaseolus vulgaris)

Plant pathogens usually promote pathogenesis by secreting effector proteins into host plant cells. One of the secreted effectors of Pseudomonas syringae pv. phaseolicola, the causative agent of halo-blight disease in common bean (Phaseolus vulgaris), HopF1, activates effector-triggered immunity (ETI) in a bean cultivar containing R1 resistance gene, but displays virulence function in a bean cultivar without the R1 gene. The virulence mechanism of the effector remained unknown, although it was identified more than a decade ago. Here we demonstrated that HopF1 can inhibit pathogen-associated molecular pattern-triggered immunity (PTI) in a susceptible bean cultivar Tendergreen. HopF1 directly interacted with two RPM1-interacting protein 4 (RIN4) orthologs of bean, PvRIN4a and PvRIN4b. Like RIN4 in Arabidopsis, both PvRIN4 orthologs negatively regulated the PTI responses in bean. However, the virulence function of HopF1 was enhanced in Tendergreen silencing PvRIN4. Furthermore, silencing PvRIN4a compromised the avrβ1-induced hypersensitive response (HR), which previously was reported to be suppressed by HopF1. Together, these results demonstrated that PvRIN4 orthologs were not the virulence target of HopF1 for inhibiting PTI, but probably for interfering with ETI.     

Genetic organization of the hrp gene cluster in Acidovorax avenae strain N1141 and a novel effector protein that elicits immune responses in rice (Oryza sativa L.)

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.     

条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

CPR5 positively regulates pattern-triggered immunity via a mediator protein

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity(PTI)and effector-triggered immunity(ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors(NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5(CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI re...     

Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys?

The phytopathogenic bacterium Pseudomonas syringae can suppress both pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) by the injection of type III effector (T3E) proteins into host cells. T3Es achieve immune suppression using a variety of strategies including interference with immune receptor signaling, blocking RNA pathways and vesicle trafficking, and altering organelle function. T3Es can be recognized indirectly by resistance proteins monitoring specific T3E targets resulting in ETI. It is presently unclear whether the monitored targets represent bona fide virulence targets or guarded decoys. Extensive overlap between PTI and ETI signaling suggests that T3Es may suppress both pathways through common targets and by possessing multiple activities.     

The Phytophthora effector Avh241 interacts with host NDR1-like proteins to manipulate plant immunity

Plant pathogens rely on effector proteins to suppress host innate immune responses and facilitate colonization. Although the Phytophthora sojae RxLR effector Avh241 promotes Phytophthora infection, the molecular basis of Avh241 virulence remains poorly understood. Here we identified non-race specific disease resistance 1 (NDR1)-like proteins, the critical components in plant effector-triggered immunity (ETI) responses, as host targets of Avh241. Avh241 interacts with NDR1 in the plasma membrane and suppresses NDR1-participated ETI responses. Silencing of GmNDR1s increases the susceptibility of soybean to P. sojae infection, and overexpression of GmNDR1s reduces infection, which supports its positive role in plant immunity against P. sojae. Furthermore, we demonstrate that GmNDR1 interacts with itself, and Avh241 probably disrupts the self-association of GmNDR1. These data highlight an effective counter-defense mechanism by which a Phytophthora effector suppresses plant immune responses, likely by disturbing the function of NDR1 during infection.     

A conserved RxLR effector interacts with host RABA‐type GTPases to inhibit vesicle‐mediated secretion of antimicrobial proteins

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co‐immunoprecipitation (Co‐IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co‐IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co‐localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH‐sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR‐1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase‐mediated vesicular secretion of antimicrobial PR‐1, PDF1.2 and possibly other defence‐related compounds.     

Recent advances in plant immunity: recognition, signaling, response, and evolution

Innate immune system is employed by plants to defend against phytopathogenic microbes through specific perception of non-self molecules and subsequent initiation of resistance responses. Current researches elucidate that plants mostly rely on cell surface-located pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NB-LRRs) to recognize pathogen-associated molecular patterns (PAMPs) and effector proteins from microbial pathogens, initiating PAMP- and effector-triggered immunity (PTI and ETI), respectively. Some pathogenic bacterial effector proteins are usually secreted into plant cells and play a virulence function by suppressing plant PTI, implying an evolutionary process of plant immunity from PTI to ETI. In the past several years, a great progress has been achieved to reveal fascinating molecular mechanisms underlying the pathogenic recognition, resistance signaling transduction, and plant immunity evolution. Here, we summarized the latest breakthroughs about these topics, and offered an integral understanding of plant molecular immunity.     

Dynamics of Defense Responses and Cell Fate Change during Arabidopsis-Pseudomonas syringae Interactions

Plant-pathogen interactions involve sophisticated action and counteraction strategies from both parties. Plants can recognize pathogen derived molecules, such as conserved pathogen associated molecular patterns (PAMPs) and effector proteins, and subsequently activate PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, pathogens can evade such recognitions and suppress host immunity with effectors, causing effector-triggered susceptibility (ETS). The differences among PTI, ETS, and ETI have not been completely understood. Toward a better understanding of PTI, ETS, and ETI, we systematically examined various defense-related phenotypes of Arabidopsis infected with different Pseudomonas syringae pv. maculicola ES4326 strains, using the virulence strain DG3 to induce ETS, the avirulence strain DG34 that expresses avrRpm1 (recognized by the resistance protein RPM1) to induce ETI, and HrcC^- that lacks the type three secretion system to activate PTI. We found that plants infected with different strains displayed dynamic differences in the accumulation of the defense signaling molecule salicylic acid, expression of the defense marker gene PR1, cell death formation, and accumulation/localization of the reactive oxygen species, H₂O₂. The differences between PTI, ETS, and ETI are dependent on the doses of the strains used. These data support the quantitative nature of PTI, ETS, and ETI and they also reveal qualitative differences between PTI, ETS, and ETI. Interestingly, we observed the induction of large cells in the infected leaves, most obviously with HrcC^- at later infection stages. The enlarged cells have increased DNA content, suggesting a possible activation of endoreplication. Consistent with strong induction of abnormal cell growth by HrcC^-, we found that the PTI elicitor flg22 also activates abnormal cell growth, depending on a functional flg22-receptor FLS2. Thus, our study has revealed a comprehensive picture of dynamic changes of defense phenotypes and cell fate determination during Arabidopsis-P. syringae interactions, contributing to a better understanding of plant defense mechanisms.     

水稻免疫机制研究进展

植物先天免疫主要由两部分组成：一类是通过细胞膜上的病原菌分子模式识别受体识别病原微生物表面存在的分子特征激发的免疫反应（PTI）;另一类是专化性的抗病R蛋白识别病原微生物的效应蛋白,从而激发下游的病原菌小种特异性的防卫反应过程（ETI）．随着水稻抗病信号途径中越来越多的抗病基因以及关键的调控基因被克隆和功能鉴定,同时多种水稻病原菌效应蛋白的发现,水稻抗病机理的研究也越来越深入．本文阐述了水稻的PTI,ETI及其下游参与免疫信号转导的关键性组分,从而形成一个初步的水稻免疫调控网络．     

A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis

Microbe- or host damage-derived patterns mediate activation of pattern-triggered immunity (PTI) in plants. Microbial virulence factor (effector)-triggered immunity (ETI) constitutes a second layer of plant protection against microbial attack. Various necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) produced by bacterial, oomycete and fungal microbes are phytotoxic virulence factors that exert immunogenic activities through phytotoxin-induced host cell damage. We here show that multiple cytotoxic NLPs also carry a pattern of 20 amino acid residues (nlp20) that triggers immunity-associated plant defenses and immunity to microbial infection in Arabidopsis thaliana and related plant species with similar characteristics as the prototype pattern, bacterial flagellin. Characteristic differences in flagellin and nlp20 plant responses exist however, as nlp20s fail to trigger extracellular alkalinization in Arabidopsis cell suspensions and seedling growth inhibition. Immunogenic nlp20 peptide motifs are frequently found in bacterial, oomycete and fungal NLPs. Such an unusually broad taxonomic distribution within three phylogenetic kingdoms is unprecedented among microbe-derived triggers of immune responses in either metazoans or plants. Our findings suggest that cytotoxic NLPs carrying immunogenic nlp20 motifs trigger PTI in two ways as typical patterns and by inflicting host cell damage. We further propose that conserved structures within a microbial virulence factor might have driven the emergence of a plant pattern recognition system mediating PTI. As this is reminiscent of the evolution of immune receptors mediating ETI, our findings support the idea that there is a continuum between PTI and ETI.     

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N_1-107) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N_1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co-evolution.     

水稻抗稻瘟病天然免疫机制及抗病育种新策略

稻瘟病是水稻最严重的病害之一,由子囊菌(Magnaporthe oryzae)引起。利用抗病品种是防治稻瘟病最经济、最有效的措施。近年来,稻瘟病已发展为研究植物与病原真菌分子互作机制的模式系统,在水稻与稻瘟菌互作和寄主抗性分子生物学、基因组学和蛋白组学等领域取得了一系列重要的研究成果。文章综述了近年来水稻抗稻瘟病两种天然免疫机制,即病原菌相关分子模式诱导和效应蛋白诱导的抗病机制研究的最新进展,讨论了GWAS、TALLEN、CRISPR和HIGS等基因组研究新方法和新技术在水稻抗病育种中的应用,并对目前稻瘟病抗性机制研究和抗病育种中的问题和挑战进行了探讨和展望。