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Natural Variation in Tomato Reveals Differences in the Recognition of AvrPto and AvrPtoB Effectors from pseudomonas syringae

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Molecular Plant 2016年第5期9卷 639-649页

作者： Christine M. Kraus Kathy R. Munkvold Gregory B. Martin Boyce Thompson Institute for Plant Research Ithaca NY 14853 USA Plant Pathology and Plant-Microbe Biology Section School of Integrative Plant Science Cornell University Ithaca NY 14853 USA Beckman Coulter Indianapolis IN 46268 USA

The Pto protein kinase from Solanum pimpinellifolium interacts with pseudomonas syringae effectors AvrPto or AvrPtoB to activate effector-triggered immunity. The previously solved crystal structures of the AvrPto-Pto and AvrPtoB-Pto complexes revealed that Pro binds each effector through both a shared and a unique interface. Hera we use natural variation in wild species of tomato to further investigate Pto recognition of these two effectors. One species, Solanum chmielewskU, was found to have many accessions that recognize only AvrPtoB. The Pto ortholog from one of these accessions was responsible for recognition of AvrPtoB and it differed from Solanum pimpinellifolium Pto by only 14 amino acids, including two in the AvrPto-specific interface, glutamate-49/glycine-51. Converting these two residues to those in Pro （histidine-49/valine-51） did not restore recognition of AvrPto. Subsequent experiments revealed that a single substitution of a histidine-to-aspartate at position 193 in Pto, which is not near the AvrPto- specific interface, was sufficient for conferring recognRion of AvrPto in plant cells. The reciprocal substi- tution of aspartate-to-histidine-193 in Pto abolished AvrPto recognition, confirming the importance of this residue. Our results reveal new aspects about effector recognition by Pto and demonstrate the value of using natural variation to understand the interaction between resistance proteins and pathogen effectors.

关键词： Effector-triggered immunity natural variation plant immunity pseudomonas syringae Pto tomato

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Role of Coronatine in Inducing Infection and Improving Resistance to pseudomonas syringae pv.mori in Mulberry Seedlings

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Agricultural Biotechnology 2017年第1期6卷 23-30,33页

作者： Na WANG Fu'an WU College of Biotechnology Jiangsu University of Science and TechnologyZhenjiang 212018China Sericulture Research Institute Chinese Academy of Agricultural SciencesZhenjiang 212018China

Mulberry bacterial blight is caused by pseudomonas syringae pv. mori. Coronatine （COR）, a phytotoxin and phytohormone produced by several strains of pseudomonas syringae, is suggested to have an important role in pathogen-plant interaction. The aim of our study was to examine the influence of COR on mul- berry in the process of pathogen infection. Results showed that COR could suppress stomatal closure induced by pathogen-associated molecular patterns （PAMPS）, assist pathogenic bacteria into the leaves, and promote bacterial proliferation in the tissues. High-concentration （1 μmol/L） exogenous COR and COR-producing bacterial pathogen induced chlorosis symptom and decrease of chlorophyll content, contrary to the effects of low-concentration （0.001 μmol/L） exogenous COR and non-COR-preducing bacterial pathogen. Treatments with COR and DC3000 enhanced the production of reactive oxygen species （ ROS）, namely, hydrogen peroxide （H2O2 ） and superexide anion （O2-）, but there were two H2O2 peaks at 1 -3 hpi and 8 -24 hpi in the DC3000-treated leaves while only one peak at 1 -3 h was observed 1 -3 h in the COR-treated ones. H202 could kill the pathogenic bacteria, on the other hand, it also acted as an upstream signaling molecule to promote nitric oxide （NO） production to further participate in the signaling pathway. Enzymatic antioxidant systems （superoxide dismutase, peroxidase and catalase） and defensive enzyme systems （lipoxygenase, polyphenol oxidase and phenylalnine ammonialyase） were activated by COR. Therefore, COR could cooperate on the inva- sion and proliferation of COR-producing bacterial pathogens, and induce the chloresis symptom in mulberry. At the same time, exogenously applied COR also could enhance the resistance to P. syringae pv. mori by production of signal molecules to activate signaling pathway and promoting defense-related metabolism.

关键词： pseudomonas syringae Coronatine Mulberry Stoma Reactive oxygen Enzyme activity

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Microbulbifer sp. ALW1和pseudomonas syringae褐藻胶裂解酶的研究

Microbulbifer sp. ALW1和Pseudomonas syringae褐藻胶裂解酶的研...

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作者：吴丽云集美大学

学位级别：硕士

本论文以从腐烂海带样品中分离得到的一株褐藻胶降解菌ALW1为研究对象,从分子和生理生化水平对其进行分类鉴定;利用硫酸铵沉淀、阴离子层析及凝胶过滤层析分离纯化获得电泳纯胞外褐藻胶裂解酶,并研究了菌株ALW1褐藻胶裂解酶的酶学性质... 详细信息

本论文以从腐烂海带样品中分离得到的一株褐藻胶降解菌ALW1为研究对象,从分子和生理生化水平对其进行分类鉴定;利用硫酸铵沉淀、阴离子层析及凝胶过滤层析分离纯化获得电泳纯胞外褐藻胶裂解酶,并研究了菌株ALW1褐藻胶裂解酶的酶学性质及其酶解产物的抗氧化活性。此外,进行了pseudomonas syringae褐藻胶裂解酶的克隆、表达、纯化、重组酶的酶学性质及其酶解产物的抗氧化活性研究,主要结论如下:(1)16S rRNA基因序列分析显示菌株ALW1归属于Microbulbifer sp.,命名为Microbulbifer sp.ALW1。结合菌株的表型及生理生化鉴定,菌株ALW1可能是Microbulbifer sp.的一个潜在新种。(2)分离纯化获得电泳纯Microbulbifer sp.ALW1褐藻胶裂解酶。底物特异性研究结果显示,该褐藻胶裂解酶同时具有降解聚甘露糖醛酸和聚古罗糖醛酸的活性。酶的最适反应温度和pH值分别为45°C和7.0,该酶在温度低于40°C,pH值为5.0-9.0时稳定。Na可明显增强褐藻胶裂解酶的活力,Ba、Ni、Zn、Co、Cu、Fe和Al对酶具有不同程度的抑制作用。除下EDTA,该褐藻胶裂解酶对检测的其他抑制剂和去垢剂具有较好的抗性。(3)Microbulbifer sp.ALW1褐藻胶裂解酶酶解产物具有良好的抗氧化活性,对ABTS和·OH自由基的半数抑制剂量IC分别为1.23 mg/mL和3.35 mg/mL,还具有还原能力。(4)pseudomonas syringae褐藻胶裂解酶的基因大小为1137 bp,预测编码含378个氨基酸残基的蛋白质。该蛋白质序列与其他菌株来源的褐藻胶裂解酶具有高的相似性,预测本研究克隆的基因编码褐藻胶裂解酶。该重组酶的理论分子量大小为42.5 kDa,理论等电点为8.15。采用同源建模法建立pseudomonas syringae褐藻胶裂解酶的三维结构,富含螺旋结构。(5)将pseudomonas syringae褐藻胶裂解酶基因进行异源表达和重组酶纯化。底物特异性研究表明,该重组酶专一性降解聚甘露糖醛酸。该重组酶的最适反应温度和pH分别为30°C和7.0,在温度低于50°C,pH值为3.0-11.0时稳定。K、Na、Li和Ca对重组酶活性没有影响,Ba、Ni、Zn、Co、Cu、Fe和Mn对重组酶具有不同程度的抑制作用。除下SDS和DTT,重组酶对检测的其他抑制剂和去垢剂具有较好的抗性。(6)pseudomonas syringae重组酶的酶解产物具有良好的抗氧化活性,对ABTS和·OH自由基的半数抑制剂量IC分别为1.56 mg/mL和0.56 mg/mL,还具有还原能力。本论文研究结果表明,Microbulbifer sp.ALW1是一株新型产胞外褐藻胶裂解酶菌株,为该菌株褐藻胶裂解酶的深入研究及活性褐藻胶寡糖的开发奠定了基础。pseudomonas syringae褐藻胶裂解酶的克隆、表达和酶学性质研究为进一步研究该酶的结构、功能及酶的定向进化奠定了良好的基础。

关键词： Microbulbifer sp. pseudomonas syringae 褐藻胶裂解酶酶学性质抗氧化活性

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Understanding plant immunity:a case study of Arabidopsis-pseudomonas syringae interaction

Understanding plant immunity:a case study of Arabidopsis-Pse...

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中国遗传学会第九次全国会员代表大会暨学术研讨会

作者： Jian-Min Zhou Institute of Genetics and Developmental Biology CAS

Plants are equipped with a variety of immune receptors controlling a sophisticated signal transduction network.They form a powerful surveillance system allowing the detection of potentially harmful microbes and rapid activation of plant defenses which culminate in physiological changes and chemical alterations that deter pathogen entry and propagation in plant tissues or the elimination of the pathogen.However,pathogenic microbes possess specialized machinery and virulence factors and are able to overcome plant immunity.Thus plant disease resistance and susceptibility is an outcome of complex interactions between plants and pathogenic microbes.A thorough understanding of plant disease resistance requires combined efforts in the study of plant immune signaling,pathogen virulence,plant physiology,and plant chemistry.We have been using the Arabidopsis-pseudomonas syringae model system to understand different facets of host pathogen interactions.I will discuss how different classes of immune receptors recognize specialized pathogens,how pathogens have evolved mechanisms to block host perception of microbes.I will also discuss how plants regulate stomatal closure to limit bacterial entry and how an adapted bacterial pathogen interferes with these processes to gain upper hand during the infection.Finally,I will report our effort in the identification of plant secondary metabolites to inhibit bacterial pathogenesis.

关键词： Plant Disease Resistance innate immunity pseudomonas syringae

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Robust transcriptional indicators of immune cell death revealed by spatiotemporal transcriptome analyses

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Molecular Plant 2022年第6期15卷 1059-1075页

作者： Jose Salguero-Linares Irene Serrano Nerea Ruiz-Solani Marta Salas-Gómez Ujjal Jyoti Phukan Victor Manuel González MartíBernardo-Faura Marc Valls David Rengel Nuria S.Coll Centre for Research in Agricultural Genomics(CRAG) CSIC-IRTA-UAB-UBCampus UABBellaterra08193 BarcelonaSpain LIPM Universitéde ToulouseINRACNRS84195 Castanet-TolosanFrance INRAE GeT-PlaGeGenotoul31326 Castanet-TolosanFrance Department of Genetics Universitat de Barcelona08028 BarcelonaSpain Consejo Superior de Investigaciones Científicas(CSIC) BarcelonaSpain Department of Plant Molecular Biology and Physiology.Albrechtvon Haller Institute for Plant Sciences niversity of GottingenJulia-Lermontowa-Weg3.37077 GottingenGrmany Institut de Phammacologie et de Biologie Structurale IPBS Universite deToulouse CNRSUPSBP 64182205 route de Nartxonne310077 Toulouse Cedex 04France

Recognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response(HR).This type of cell death occurs precisely at the site of pathogen recognition,and it is restricted to a few cells.Extensive research has shed light on how plant immune receptors are mechanistically activated.However,two central key questions remain largely unresolved:how does cell death zonation take place,and what are the mechanisms that underpin this phenomenon?Consequently,bona fide transcriptional indicators of HR are lacking,which prevents deeper insight into its mechanisms before cell death becomes macroscopic and precludes early or live observation.In this study,to identify the transcriptional indicators of HR we used the paradigmatic Arabidopsis thaliana–pseudomonas syringae pathosystem and performed a spatiotemporally resolved gene expression analysis that compared infected cells that will undergo HR upon pathogen recognition with bystander cells that will stay alive and activate immunity.Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes,expression profiles,and biological processes derived from tissue undergoing HR and that of its surroundings.Furthermore,we generated a pipeline based on concatenated pairwise comparisons between time,zone,and treatment that enabled us to define 13 robust transcriptional HR markers.Among these genes,the promoter of an uncharacterized AAA-ATPase was used to obtain a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death.This valuable set of genes can be used to define cells that are destined to die upon infection with HR-triggering bacteria,opening new avenues for specific and/or high-throughput techniques to study HR processes at a single-cell level.

关键词： Arabidopsis thaliana cell death indicator effector-triggered immunity hypersensitive response pattern-triggered immunity plant immunity pseudomonas syringae

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Circadian Clock-Regulated Phosphate Transporter PHT4;1 Plays an Important Role in Arabidopsis Defense

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Molecular Plant 2011年第3期4卷 516-526页

作者： Guo-Ying Wang Jiang-Li Shi Gina Ng Stephanie L. Battle Chong Zhang Hua Lu Department of Biological Sciences University of Maryland Baltimore County 1000 Hilltop CircLe Baltimore MD 21250 USA

The Arabidopsis accelerated cell death 6-1 （acd6-1） mutant shows constitutive defense, cell death, and ex- treme dwarf phenotypes. In a screen for acd6-1 suppressors, we identified a mutant that was disrupted by a T-DNA in the PHOSPHATE TRANSPORTER 4;1 （PHT4;1） gene. The suppressor mutant pht4;1-1 is dominant, expresses truncated PHT4;1 transcripts, and is more susceptible to virulent pseudomonas syringae strains but not to several avirulent strains. Treat- ment with a salicylic acid （SA） agonist induced a similar level of resistance in Col-0 and pht4;1-1, suggesting that PHT4;1 acts upstream of the SA pathway. Genetic analysis further indicates that PHT4,1 contributes to SID2-dependent and -in- dependent pathways. Transgenic expression of the DNA fragment containing the PHT4;1-1 region or the full-length PHT4;1 gene in wild-type conferred enhanced susceptibility to pseudomonas infection. Interestingly, expression of PHT4;1 is reg- ulated by the circadian clock. Together, these data suggest that the phosphate transporter PHT4;1 is critical for basal defense and also implicate a potential role of the circadian clock in regulating innate immunity of Arabidopsis.

关键词： Biological clock disease resistance signal transduction pseudomonas syringae phosphate transporter salicylic acid.

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Regulatory and Functional Aspects of Indolic Metabolism in Plant Systemic Acquired Resistance

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Molecular Plant 2016年第5期9卷 662-681页

作者： Elia Stahl Patricia Bellwon Stefan Huber Klaus Schlaeppi Friederike Bernsdorff Armelle Vallat-Michel Felix Mauch Jurgen Zeier Department of Biology Heinrich Heine University Dusseidorf Universiyatsstraβ 1 40225 Dusseidorf Germany Plant Biology Section University of Fribourg Route Albert Gockel 3 1700 Fribourg Switzerland Institut de Chimie Unlversite de Neuchatel Avenue Bellevaux 51 2007 Neuchatel Swttzerland Cluster of Excellence on Plant Sciences (CEPLAS) Heinrich Heine University Dusseldorf Universitatsstraβe 1 D-40225 Dusseldorf Germany Agroscope Institute for Sustainability Sciences Reckenholzstrasse 191 CH-8046 Zurich Switzerland

Tryptophan-derived, indolic metabolites possess diverse functions in Arabidopsis innate immunity to microbial pathogen infection. Hers, we investigate the functional role and regulatory characteristics of indolic metabolism in Arabidopsis systemic acquired resistance （SAR） triggered by the bacterial pathogen pseudomonas syringae. Indolic metabolism is broadly activated in both P. syringae-inoculated and distant, non-inoculated leaves. At inoculation sites, camalexin, indol-3-ylmethylamine （13A）, and indole-3-carboxylic acid （ICA） are the major accumulating compounds. Camalexin accumulation is positively affected by MYB122, and the cytochrome P450 genes CYP81F1 and CYP81F2. Local 13A production, by contrast, occurs via indole glucosinolate breakdown by PEN2- dependent and independent pathways. Moreover, exogenous application of the defense hormone salicylic acid stimulates 13A generation at the expense of its precursor indol-3-ylmethylglucosinolate （13M）, and the SAR regulator pipecolic acid primes plants for enhanced P. syringae-induced activation of distinct branches of indolic metabolism. In uninfected systemic tissue, the metabolic response is more specific and associated with enhanced levels of the indolics 13A, ICA, and indole-3-carbaldehyde （ICC）. Systemic indole accumulation fully depends on functional CYP79B2/3, PEN2, and MYB34/51/122, and requires functional SAR signaling. Genetic analyses suggest that systemi- cally elevated indoles are dispensable for SAR and associated systemic increases of salicylic acid. However, soil-grown but not hydroponically -cultivated cyp79b2/3 and pen2 plants, both defective in indolic secondary metabolism, exhibit pre-induced immunity, which abrogates their intrinsic ability to induce SAR.

关键词： indolic metabolism systemic acquired resistance pseudomonas syringae Arabidopsis thaliana

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Antagonism of Transcription Factor MYC2 by EDS1/PAD4 Complexes Bolsters Salicylic Acid Defense in Arabidopsis Effector-Triggered Immunity

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Molecular Plant 2018年第8期11卷 1053-1066页

作者： Haitao Cui Jingde Qiu Yue Zhou Deepak D. Bhandari Chunhui Zhao Jaqueline Bautor Jane E. Parker Department of Plant-Microbe Interactions Max-Planck Institute for Plant Breeding Research Carl-von-Linne Weg 10 50829 Cologne Germany Department of Plant Developmental Biology Max-Planck Institute for Plant Breeding Research 50829 Cologne Germany Key Laboratory of Ministry of Education for Genetics Breeding and Multiple Utilization of Crops Plant Immunity Center Fujian Agriculture University Fuzhou 350002 China These authors contributed equaily to this article.

In plant immunity, pathogen-activated intracellular nucleotide binding/leucine rich repeat （NLR） receptors mobilize disease resistance pathways, but the downstream signaling mechanisms remain obscure. Enhanced disease susceptibility 1 （EDS1） controls transcriptional reprogramming in resistance triggered by Toll-lnterleukinl-Receptor domain （TIR）-family NLRs （TNLs）. Transcriptional induction of the salicylic acid （SA） hormone defense sector provides one crucial barrier against biotrophic pathogens. Here, we present genetic and molecular evidence that in Arabidopsis an EDS1 complex with its partner PAD4 inhibits MYC2, a master regulator of SA-antagonizing jasmonic acid （JA） hormone pathways. In the TNL immune response, EDSl/PAD4 interference with MYC2 boosts the SA defense sector independently of EDS1-induced SA synthesis, thereby effectively blocking actions of a potent bacterial JA mimic, coronatine （COR）. We show that antagonism of MYC2 occurs after COR has been sensed inside the nucleus but before or coincident with MYC2 binding to a target promoter, pANAC019. The stable interaction of PAD4 with MYC2 in planta is competed by EDS1-PAD4 complexes. However, suppression of MYC2-promoted genes requires EDS1 together with PAD4, pointing to an essential EDS1-PAD4 heterodimer activity in MYC2 inhibition. Taken together, these results uncover an immune receptor signaling circuit that intersects with hormone pathway crosstalk to reduce bacterial pathogen growth.

关键词： NLR receptor stress hormone network pseudomonas syringae AvrRps4 RRS1/RPS4 coi1

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MYC2： The Master in Action

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Molecular Plant 2013年第3期6卷 686-703页

作者： Kemal Kazan John M. Manners Commonwealth Scientific and Industrial Research Organization (CSIRO) Plant Industry Queensland Bioscience Precinct Brisbane Queensland 4067 Australia

Jasmonates （JAs） are plant hormones with essential roles in plant defense and development. The basic- helix-loop-helix （bHLH） transcription factor （TF） MYC2 has recently emerged as a master regulator of most aspects of the jasmonate （JA） signaling pathway in Arabidopsis. MYC2 coordinates JA-mediated defense responses by antagonistically regulating two different branches of the JA signaling pathway that determine resistance to pests and pathogens, respectively. MYC2 is required for induced systemic resistance （ISR） triggered by beneficial soil microbes while MYC2 function is targeted by pathogens during effector-mediated suppression of innate immunity in roots. Another notable function of MYC2 is the regulation of crosstalk between the signaling pathways of JA and those of other phytohormones such as abscisic acid （ABA）, salicylic acid （SA）, gibberellins （GAs）, and auxin （IAA）. MYC2 also regulates interactions between JA signaling and light, phytochrome signaling, and the circadian clock, MYC2 is involved in JA-regulated plant development, lateral and adventitious root formation, flowering time, and shade avoidance syndrome. Related bHLH TFs MYC3 and MYC4 also regulate both overlapping and distinct MYC2-regulated functions in Arabidopsis while MYC2 orthologs act as ＇master switches＇ that regulate JA-mediated biosynthesis of secondary metabolites. Here, we briefly review recent studies that revealed mechanistic new insights into the mode of action of this versatile TF.

关键词： ABA auxin DELLAs gibberellin hormonal crosstalk jasmonate JAZ proteins light signaling MED25 MYC2 plant defense phytochrome pseudomonas syringae salicylic acid.

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A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

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Plant Communications 2022年第1期3卷 6-20页

作者： Ayumi Matsumoto Titus Schluter Katharina Melkonian Atsushi Takeda Hirofumi Nakagami Akira Mine Research Organization of Science and Technology Ritsumeikan UniversityShiga 525-8577Japan Basic Immune System of Plants Max Planck Institute for Plant Breeding ResearchCologne 50829Germany College of Life Sciences Ritsumeikan UniversityShiga 525-8577Japan JST PRESTO Kawaguchi-shiSaitama 332-0012Japan Laboratory of Plant Pathology Graduate School of AgricultureKyoto UniversityKyoto 606-8502Japan

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays.Here,we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants.We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABEluciferase operon into a specific genomic location found ubiquitously across bacterial phyla.These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera pseudomonas,Rhizobium(Agrobacterium),and Ralstonia.Direct luminescence measurements of plant tissues inoculated with bioluminescent pseudomonas syringae pv.tomato DC3000(Pto-lux)reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana,Solanum lycopersicum,Nicotiana benthamiana,and Marchantia polymorpha.We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains.Importantly,quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth,with a dynamic range of four orders of magnitude.Moreover,macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues.In conclusion,our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions.

关键词： plant immunity bacterial virulence bioluminescence imaging pseudomonas syringae Arabidopsis thaliana Marchantia polymorpha

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