寄主植物与病原菌免疫反应的分子遗传基础

近年来,大量的植物抗病基因和病原菌无毒基因被克隆,抗病基因和无毒基因的结构、功能及其互作关系的研究也取得重大进展。在植物中,由病原菌模式分子(pathogen-associated molecular patterns, PAMPs)引发的免疫反应(PAMP-triggered immunity, PTI)和由效应因子引发的免疫反应(effector-triggered immunity, ETI)是植物在长期进化过程中形成的两类抵抗病原物的机制。PTI反应主要通过细胞表面受体(patternrecognition receptors, PRRs)识别并结合PAMPs从而激活下游免疫反应,而在ETI反应中,则通过植物R基因(resistance gene,R)与病原菌无毒基因(avirulence gene, Avr)产物间的直接或间接相互作用来完成免疫反应。本文对植物PTI反应和ETI反应分别进行了概述,重点探讨了植物R基因与病原菌Avr基因之间的互作遗传机理,并对目前植物抗性分子遗传机制研究和抗病育种中的问题进行了探讨和展望。     

Identification of MAPKs and their possible MAPK kinase activators involved in the Pto-mediated defense response of tomato

The Pto kinase mediates resistance to bacterial speck disease in tomato by activating host defenses upon recognition of Pseudomonas syringae pv. tomato strains expressing the AvrPto or AvrPtoB proteins. Previous gene-silencing experiments have indicated that mitogen activated protein kinase (MAPK) cascades play a key role downstream of the Pto kinase to activate host defense responses. Here we use biochemical methods to demonstrate that two tomato MAPKs, LeMPK2 and LeMPK3, are activated in leaves in a Pto-specific manner upon expression of AvrPto and AvrPtoB. We show that these same MAPKs are activated upon overexpression of LeMAPKKKalpha, a protein previously demonstrated to be involved in Pto-mediated immunity. We identified two phylogenetically unrelated MAPK kinases (LeMKK2 and LeMKK4) that when overexpressed in leaves elicit cell death and activate LeMPK2 and LeMPK3. In vitro analysis demonstrated that LeMKK2 and LeMKK4 each phosphorylate the same subset of three MAPKs. Together these data provide biochemical evidence for the involvement of MAPK cascades in Pto-mediated resistance.     

Plant immunity: Evolutionary insights from PBS1, Pto and RIN4

Two layers of plant immune systems are used by plants to defend against phytopathogens. The first layer is pathogen-associate molecular patterns (PAMPs)-triggered immunity (PTI), which is activated by plant cell-surface pattern recognition receptors (PRRs) upon perception of microbe general elicitors. The second layer is effector-triggered immunity (ETI), which is initiated by specific recognition of pathogen type III secreted effectors (T3SEs) with plant intracellular resistance (R) proteins. Current opinions agree that ETI was evolved from PTI, and the impetus for the evolution of plant immunity is pathogen T3SEs, which exhibit virulence functions through blocking PTI, but show avirulence functions for triggering ETI. A decoy model was put forward and explained that the avirulence targets of pathogen T3SEs were evolved as decoys to compete with the virulence targets for binding with pathogen T3SEs. However, little direct evidence for the evolutionary mode has been offered. Here we reviewed the recent progresses about Pto, PBS1 and RIN4 to present our viewpoints about the evolution of plant immunity.Key words: plant immunity, evolution, Pto, PBS1, RIN4     

植物与病原菌互作的分子机制研究进展

植物的先天免疫主要包括模式识别受体对保守的微生物病原相关分子模式的识别和抗病蛋白对效应蛋白的识别。植物与病原体互作过程中存在广泛的信号交流,信号分子在植物与病原体的互作攻防中发挥了重要的调控作用,决定了二者的竞争关系。当前,大量植物与病原体互作中的信号分子被定位和克隆,其作用方式被揭示。本文总结了这些信号分子及其在植物免疫过程中的作用机制,主要包括植物细胞表面的模式识别受体分子对病原相关分子模式的识别与应答,植物抗病蛋白对病原体效应蛋白的识别与应答,以及免疫反应下游相关信号分子及其在植物抗病中的作用。此外,本文对未来相关研究提出了展望。     

Tight regulation of plant immune responses by combining promoter and suicide exon elements

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.     

Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

Prf immune complexes of tomato are oligomeric and contain multiple Pto‐like kinases that diversify effector recognition

Cytoplasmic recognition of pathogen virulence effectors by plant NB‐LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB‐LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N‐terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N‐terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non‐Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

Pseudomonas syringae type III effector AvrPtoB is phosphorylated in plant cells on serine 258, promoting its virulence activity

The Pseudomonas syringae pv. tomato protein AvrPtoB is translocated into plant cells via the bacterial type III secretion system. In resistant tomato leaves, AvrPtoB acts as an avirulence protein by interacting with the host Pto kinase and eliciting the host immune response. Pto-mediated immunity requires Prf, a Pto-interacting protein with a putative nucleotide-binding site and a region of leucine-rich repeats. In susceptible tomato plants, which lack either Pto or Prf, AvrPtoB acts as a virulence protein by promoting P. syringae pv. tomato growth and enhancing symptoms associated with bacterial speck disease. The N-terminal 307 amino acids of AvrPtoB (designated AvrPtoB(1-307)) are sufficient for these virulence activities and for Pto-mediated avirulence. We report that AvrPtoB is phosphorylated by a Pto- and Prf-independent kinase activity that is conserved in several plant species, including tomato (Solanum lycopersicum), Nicotiana benthamiana, and Arabidopsis thaliana. AvrPtoB(1-307) was phosphorylated in tomato protoplasts, and mass spectrometry identified serine 258 as the major in vivo phosphorylation site of this protein. An alanine substitution of Ser(258) resulted in the loss of virulence and the diminution of avirulence activity of AvrPtoB(1-307), whereas a phosphomimetic S258D mutant had activities similar to wild type AvrPtoB(1-307). These observations suggest that AvrPtoB has evolved to mimic a substrate of a conserved plant kinase, leading to enhancement of its virulence and avirulence activities in the host cell.     

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.     

Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins

Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction.     

Role of pathogen's effectors in understanding host-pathogen interaction

Pathogens can pose challenges to plant growth and development at various stages of their life cycle. Two interconnected defense strategies prevent the growth of pathogens in plants, i.e., molecular patterns triggered immunity (PTI) and pathogenic effector-triggered immunity (ETI) that often provides resistance when PTI no longer functions as a result of pathogenic effectors. Plants may trigger an ETI defense response by directly or indirectly detecting pathogen effectors via their resistance proteins. A typical resistance protein is a nucleotide-binding receptor with leucine-rich sequences (NLRs) that undergo structural changes as they recognize their effectors and form associations with other NLRs. As a result of dimerization or oligomerization, downstream components activate “helper” NLRs, resulting in a response to ETI. It was thought that ETI is highly dependent on PTI. However, recent studies have found that ETI and PTI have symbiotic crosstalk, and both work together to create a robust system of plant defense. In this article, we have summarized the recent advances in understanding the plant's early immune response, its components, and how they cooperate in innate defense mechanisms. Moreover, we have provided the current perspective on engineering strategies for crop protection based on up-to-date knowledge.     

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.     

HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens

? Plant immunity is activated by sensing either conserved microbial signatures, called pathogen/microbe-associated molecular patterns (P/MAMPs), or specific effectors secreted by pathogens. However, it is not known why most microbes are nonpathogenic in most plant species. ? Nonhost resistance (NHR) consists of multiple layers of innate immunity and protects plants from the vast majority of potentially pathogenic microbes. Effector-triggered immunity (ETI) has been implicated in race-specific disease resistance. However, the role of ETI in NHR is unclear. ? Pseudomonas syringae pv. tomato (Pto) T1 is pathogenic in tomato (Solanum lycopersicum) yet nonpathogenic in Arabidopsis. Here, we show that, in addition to the type III secretion system (T3SS)-dependent effector (T3SE) avrRpt2, a second T3SE of Pto T1, hopAS1, triggers ETI in nonhost Arabidopsis. ? hopAS1 is broadly present in P. syringae strains, contributes to virulence in tomato, and is quantitatively required for Arabidopsis NHR to Pto T1. Strikingly, all tested P. syringae strains that are pathogenic in Arabidopsis carry truncated hopAS1 variants of forms, demonstrating that HopAS1-triggered immunity plays an important role in Arabidopsis NHR to a broad-range of P. syringae strains.     

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.     

Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.