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茄青枯病菌Rs-T02 RSc3316基因功能初步分析

茄青枯病菌Rs-T02 RSc3316基因功能初步分析

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者：汪锴豪张锡娇李凤芳袁高庆林纬黎起秦广西大学农学院

植物青枯病（bacterial wilt）是由茄青枯拉尔氏菌（Ralstonia solanacearum Yabuuchi et al.）引起的植物土传病害,严重威胁作物的生产。本研究的前期工作发现,在3,4,5-三羟基苯甲酸甲酯（methyl gallate,简称MG）胁迫下,茄青枯拉尔氏... 详细信息

植物青枯病（bacterial wilt）是由茄青枯拉尔氏菌（Ralstonia solanacearum Yabuuchi et al.）引起的植物土传病害,严重威胁作物的生产。本研究的前期工作发现,在3,4,5-三羟基苯甲酸甲酯（methyl gallate,简称MG）胁迫下,茄青枯拉尔氏菌Rs-T02菌株的转录组差异分析表达上调的FF-ATP合酶ε亚基蛋白（FF-ATP synthase subunit epsilon）基因（Gene ID:GM001812）,其编码蛋白的氨基酸序列与差异蛋白组分析新增的蛋白（Accession number:gi17548033）的氨基酸序列一致,编码该蛋白的基因为RSc3316,且在MG作用下的Rs-T02菌株生长受阻,致病力下降。我们推出FF-ATP合酶ε亚基可能是MG作用茄青枯拉尔氏菌的靶标之一。为了探索该菌中的FF-ATP合酶ε亚基基因（RSc3316）的功能,本研究以Rs-T02野生菌R、FF-ATP合酶ε亚基（RSc3316）缺失突变体菌N及互补菌C为试验菌株,用注射接种法测定其对番茄的致病力,用双抗体夹心法酶联免疫吸附试验（ELISA）测定其胞外纤维素酶活性,利用果胶酶水解果胶生成半乳糖醛酸法测定其胞外果胶酶活性,采用苯酚-硫酸法进行比色测定其胞外多糖含量,通过测定Pi增加速率测定其FF-ATP合酶活性,为进一步研究MG的抑菌机制提供依据。研究结果显示,用注射法将10μL菌体浓度为5×10～7CFU/mL的野生菌R、RSc3316基因缺失突变体菌N和接种互补菌C分别接种到番茄苗从上往下的第2个腋芽中,接种30d后,接种番茄植株发病率分别为100%、0和91%;在NA培养液震荡（28℃、130min）培养24 h、菌体数量为1×10～9CFU/mL的野生菌R、RSc3316基因缺失突变体菌N和互补菌C的胞外纤维素酶活性分别为32.49U/mL、11.29U/mL和26.39U/mL,胞外果胶酶活性分别为5.43mg/（h·mL）、5.35mg/（h·mL）和0.96mg/（h·mL）,产生胞外多糖的量分别为2.41μg、2.42μg和1.84μg,胞内FF-ATP酶活性分别为0.73μmol/min/10～9CFU、1.19μmol/min/10～9CFU和0.87μmol/min/10～9GEU。研究结果表明,Rs-T02菌株缺失RSc3316基因后菌体中纤维素酶和果胶酶的活性下降、产胞外多糖的能力降低,F01ATP酶的活性增加,而对番茄的致病力下降。

关键词：茄青枯病菌 RSc3316基因功能 3,4,5-三羟基苯甲酸甲酯

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The bHLH transcription factor PUIP2 regulates plant immunity,senescence and development in Arabidopsis thaliana

The bHLH transcription factor PUIP2 regulates plant immunity...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者： Huang Zhe Liu Jing Li Wei Dai Liangying College of Plant Protection Hunan Agricultural University

The transcription factors are vital for many important cellular processes in plant,for instance,cell cycle,response to environment,development,disease resistance,and so *** we show that PUIP2,a typical bHLH transcription factor from Arabidopsis thaliana,was identified as a regulator of both flagellin and chitin triggered immunity(i.e.P AMP-triggered immunity,PTI).The transcription level of PUIP2 is enhanced obviously after Arabidopsis thaliana being treated with the PAMP flagellin and chitin,***,the PUIP2 is also activated by the inoculation of pathogens Pseudomonas syringae *** DC3000 and Powdery ***,we treated the PUIP2 T-DNA knock-out mutant with flagellin,chitin,***3000 and *** respectively,and found that the puip2 mutant shows decreased PTI responses and compromised disease resistance against ***3000 and ***,the transcription level of the resistance related genes,such as PR1 and NPR1,is inhibited in the puip2 *** the planting of the puip2 mutant,we found that the lower leaves of puip1 display much less chlorosis(***) dian wild-type under long day(16 h light/8 h dark) ***,we found the dexamethasone-induced cell death is also inhibited in puip2 mutant,while the PUIP2 over expression transgenic plants display spontaneous cell death *** the other hand,the leaves and whole plant size of puip2 mutant are smaller than the wild-type ***,the hypocotyl of puip2 mutant is much longer than wild-type under blue and far red light but not white light,red light and odier *** on CHIP-seq data,we screened and identified disease resistance and senescence pathways related genes as candidate targets of PUIP2,which are needed to further *** summary,the transcription factor PUIP2 synthetically regulates plant innate immunity,senescence and development.

关键词： The bHLH transcription factor PUIP2 regulates plant immunity,senescence and development in Arabidopsis thaliana

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Brown leaf spot disease of kiwifruit in China caused by Neofusicoccum parvum

Brown leaf spot disease of kiwifruit in China caused by Neof...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者： Li Li Pan Hui Deng Lei Chen Meiyan Zhong Caihong Key Laboratory of Plant Germplasm Enement and Specialty Agriculture Wuhan Botanical GardenChinese Academy of Sciences

In August 2016,brown leaf spot disease of kiwifruit was found in orchards in Liupanshui,Guizhou Province and Taishun,Zhejiang *** appeared as foliar discolorations,such as grey to brown sectorial necrosis,with infected leaves dying while attached to the *** morphologically similar fungal isolates(NP1-10) were isolated from both *** were fusoid,hyaline,unicellular with sub-obtuse apex and truncate base,fusiform to ellipsoidal with rounded apices,truncated bases,thin walls,and a smooth *** conidia were measured and ranged from 15.43-17.58μm in length and 4.37-6.99μm in *** traits match those reported for Neofusicoccum parvum(Pennycook & Samuels) Crous,Slippers & *** rDNA internal transcribed spacer(ITS)region and β-tubulin(BT) gene were amplified using primers ITS4/ITS5 and BT2a/*** sequences(Accession ***314146-MF314155 for the ITS region;MF314156 and MF314165 for β-tubulin) were identical to *** strain MAR2134(Accession ***997474 and KU997593) reported in GenBank using BLASTn,confirmed by molecular phylogenetic trees constructed using *** species was thus identified as *** test was performed on 12 nonwounded/wounded young leaves of cultivar 'Hongyang' by inoculated conidial suspension(10μL,10～6 conidia/mL) and colonized PDA pieces(5 mm in diameter) from 7 day-old *** equal number of control plants were served with sterile PDA plugs and distilled *** inoculated plant developed typical necrotic lesions,while control and unwounded leaves remained *** was consistently re-isolated only from inoculated leaves,confirming Koch's *** has been reported to cause dieback and canker in various fruit trees,including blueberryand mango *** is the first known report of *** causing brown spot disease inkiwifruit in China.

关键词： Kiwifruit Brown Leaf Spot Disease Neofusicoccum parvum

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Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Alternaria solani

Comparative evaluation of the LAMP assay and PCR-based assay...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者： Mehran Khan Wang Rongbo Li Benjin Liu Peiqing Weng Qiyong Chen Qinghe Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests Institute of Plant ProtectionFujian Academy of Agricultural Sciences

Early blight(EB),caused by the pathogen Alternaria solani,is a major threat to global potato and tomato *** and accurate diagnosis of this disease is therefore *** this study,we conducted a loop-mediated isothermal amplification(LAMP) assay,as well as conventional polymerase chain reaction(PCR),nested PCR,and quantitative real-time PCR(RT-qPCR) assays to determine which of these techniques was less time consuming,more sensitive,and more *** based our assays on sequence-characterised amplified regions of the histidine kinase *** LAMP assay provided more rapid and accurate results,amplifying the target pathogen in less than 60 min at63℃,with 10-fold greater sensitivity than conventional *** PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional ***-qPCR was the most sensitive among the assays evaluated,being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration(100 fg).The LAMP assay was more sensitive than conventional PCR,but less sensitive than nested PCR and RT-qPCR;however,it was simpler and faster than the other assays *** LAMP assay amplified *** artificially,allowing us to naturally infect young potato leaves,which produced early symptoms of *** LAMP assay also achieved positive amplification using diluted pure *** culture instead of genomic ***,this technique has greater potential for developing quick and sensitive visual detection methods than other conventional PCR strategies for detecting *** in infected plants and culture,permitting early prediction of disease and reducing the risk of epidemics.

关键词： Alternaria solani Early blight histidine kinase gene LAMP Real-time PCR Sensitivity Specificity

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Role of G Protein Signaling pathway in N-acyl-homoserine lactones-elicited plant resistance

Role of G Protein Signaling pathway in N-acyl-homoserine lac...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者： Liu Fang Zhao Qian Huang Yali Jia Zhenhua Song Shuishan Biology Institute Hebei Academy of SciencesHebei Engineering and Technology Center of Microbiological Control on Main Crop Disease

N-acyl-homoserine lactones（AHLs） are the quorum-sensing（QS） signal molecules used to coordinate the population behaviors in gram-negative *** evidence demonstrates that plants are able to respond to bacterial AHLs,including intracellular calcium elevation and G protein-mediated AHL-induced plant root ***,little is known about the molecular mechanism of plants reacting to these bacterial *** we show that treatment of Arabidopsis roots with the N-3-oxo-octanoyl-homoserine lactone（3OC8-HSL） significantly increased plant resistance to infection of pathogen bacteria Pseudomonas syringae pv tomato *** with 3OC8-HSL and subsequent pathogen invasion triggered an augmented burst of hydrogen peroxide,accumulation of salicylic acid（SA）,and fortified expression of the Pathogen-related 1 and 5（PR1/PR5） *** increased resistance in wild type Arabidopsis induced by 3OC8-HSL were abolished in GPA1 functional-deficiency mutant gpa1-4 and AGB1 functional-deficiency mutant ***,3OC8-HSL-primed resistance to PstDC3000 in wild-type plants was impaired by GCR2 functional-deficiency mutating while enhanced in plants GCR2 *** concentration of SA and the accumulation of HO were increased by3OC8-HSL,and this effect was weakened by Gα functional-deficiency mutating and Gβ functional-deficiency *** data suggested that G protein signaling play a positive role in plant resistance induced by bacterial *** out-come of this study will be great importance of further understanding AHL-mediated cross-kingdom communication between bacteria and host plant.

关键词： Role of G Protein Signaling pathway in N-acyl-homoserine lactones-elicited plant resistance

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Maize homologs of COMT,a key enzyme in lignin biosynthesis,modulate the hypersensitive response mediated by resistance protein Rp1-D21

Maize homologs of COMT,a key enzyme in lignin biosynthesis,m...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者：刘孟洁 Sun Yang Luan Qingling Liu Xiaoying Wang Mei Zhu Yuxiu Mao Weichao 王官锋 The Key Laboratory of Plant Cell Engineering and Germplasm Innovation Ministry of EducationCollege of Life SciencesShandong University

Rp1-D21,a naturally occurring mutant in maize,derives from an intragenic recombination event between two CC-NB-LRR genes,Rp1-D and ***-D21 induces an auto-active hypersensitive response(HR) in maize and Nicotiana benthamiana in the absence of pathogen infection,which has been used as a powerful tool to investigate the genes function in modulating the HR mediated by *** plays significant roles in plant development and defense and multiple key enzymes in lignin biosynthesis pathway have been reported to be involved in response to pathogen *** our previous studies,hydroxycinnamoyltransferase(HCT) and caffeoyl-CoA O-methyltransferase(CCoAOMT),two key enzymes in lignin biosynthesis,have been proved to suppress Rpl-D21-mediated HR through physical *** acid O-methyltransferase(COMT) is another key enzyme in lignin biosynthesis and the well-known bm3(brown midrib 3) mutant in maize is due to the disruption of *** investigate the function of COMT in Rpl-D21-mediated HR,two homologs of COMT(COMT1 and COMT2) were transient co-expression with Rpl-D21 or its functional signaling domain coiled cob(CC)in *** results showed that COMT2,but not COMT1 suppressed both Rpl-D21 and CC mediated ***-IP assay showed that COMT2 interacts with CC in ***,COMT2,but not COMT1 interacts with HCT and CCoAOMT,suggesting these three proteins might form a complex in *** results indicate that some homologs of key enzymes in lignin biosynthesis may be synergetic to regulate Rp1-D21-mediated *** study will clarify the regulatory function of maize COMT homologs in defense response and it is potential to use COMT1 as a new gene resource for maize genetic improvement.

关键词： Maize Hypersensitive response Rp1-D21 COMT

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Determination of bioactivity of culture filtrates of the pathogen causing black spots on the Citrus maxima

Determination of bioactivity of culture filtrates of the pat...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者：卢松茂 Li Lirong Lu Guodong Lin Xiuxiang Fujian Institute of Tropic Crops Zhangzhou Meteorological Experiment Station of Tropic Crops Key Laboratory of Biopesticide and Chemical Biology Ministry of EducationFujian Agriculture and Forestry University

[Objective]Phyllosticta citriasiana,the causal agent of Pomelos Black Spot,affects pomelos plants throughout subtropical climates in East Asia,causing a reduction in both fruit quantity and ***,PBS is a major constraints in the production of high quality and wholesome pomelo *** investigations showed that some bioactivity compounds including tauranin,PMtoxins B and C,phyllosinol,phyllostine,phycarone,brefeldin A,cholesterol and elsinochrome A-C had been isolated from some fungal strains of the genus ***,*** had been reported that its crude extracts could inhibit the growth of Escherichia coli,Bacillus cereus,and Pseudomonas aeruginosa,but little is documented about the bioactivities of the cultural filtrates of *** on the host(Citrus maxima) *** this study,the growth morphology and growth rate of *** Pch-1 strain cultured in the different liquid media and the bioactivities of culture filtrates of Pch-1 strain on the wounded leaves of Citrus maxima *** were determined,to preliminarily understand the mechanism of Pch-1 strain infection host leaves and to help further isolation and identification phytotoxic components of Pch-1 strain.[Method]Initially,Pch-1 strain was cultured on the potato dextrose agar(PDA) and cultivated at 25℃ for 2 *** pieces of mycelial disks(5 mm) cut from margin of colony of Pch-1 strain,were kept in a flask with liquid media(100 mL),such as oat liquid medium(OLM),potatoes dextrose liquid medium(PDLM),potatoes sugar liquid medium(PSLM),potatoes sugar potassium phosphate dibasic liquid medium(PSP),improved potatoes dextrose liquid medium(IPDLM),Richard liquid medium(RLM) and Czapek-dox liquid medium(CLM)) with three replications,*** flasks were incubated on an orbital incubator at 120 r/min at 25℃ for 7 days,to investigate the mycelia growth rate and the growth morphology of Pch-1 *** the other hand,the culture filtrates of Pch-1

关键词： Pomelos black spot Phyllosticta citriasiana Culture filtrates Bioactivities

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The interaction between Turnip mosaic virus encoded proteins and SWEET proteins in Arabidopsis thaliana

The interaction between Turnip mosaic virus encoded proteins...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者： Sun Ying Sun Yue Wang Yan Pan Hongyu Zhang Xianghui Zhang Yanhua Liu Jinliang Department of Plant Protection College of Plant SciencesJilin University

Turnip mosaic virus(TuMV) is an important species of the genus Potyvirus and has an exceptionally broad host range in terms of plant genera and families of any *** occurs worldwide and causes great losses to agricultural *** symptom formation of a plant viral disease results from molecular interactions between the virus and its host *** Will Eventually be Exported Transporters(SWEET) are a novel type of sugar transporter that play crucial role in the interaction of *** has been reported that plants infected with fungi or bacteria will induce partial expression of the SWEET gene involved in pathogen-host ***,the SWEET protein family has not been found to be involved in the interaction between virus and *** this study,a yeast two hybrid library of Arabidopsis thaliana cDNAs was screened using the P3 protein as ***1 in Arabidopsis thaliana was identified to interact with TuMV P3 *** in deeply confirmed that AtSWEET1 interacted with TuMV-encoded P3,HC-Pro,VPg and NIa-Pro protein by yeast two-hybrid assay and Bimolecular fluorescence complementation assay(BiFC).In order to study whether Arabidopsis thaliana 17 AtSWEET genes were induced by virus *** expression of AtSWEET genes were detected to infect Arabidopsis thaliana by infectious cDNA clone of *** result showed that most of the AtSWEET genes were induced *** on this result,up-regulated genes encoded AtSWEET 4,7,8,9,11,15 proteins were selected to verify the interaction with TuMV encoded *** two-hybrid assay showed that AtSWEET4 interacted with TuMV-encoded P3 and NIb protein,AtSWEET7 interacted with VPg and NIb protein,AtSWEET8 interacted with P3 and NIb protein,AtSWEET1 interacted with P3,HC-Pro,PIPO,CI and NIb protein,AtSWEET11 interacted with P3,HC-Pro,PIPO,CI and NIb protein,AtSWEET15 interacted with P3 and NIb *** was in deeply confirmed by *** suggested that SWEET protein family pl

关键词： Turnip mosaic virus SWEET proteins Arabidopsis thaliana Interaction

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Maize metacaspase inhibits the hypersensitive response induced by autoactive NLR protein Rp1-D21

Maize metacaspase inhibits the hypersensitive response induc...

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者：栾庆玲 Liu Mengjie Sun Yang Wang Mei Liu Xiaoying Zhu Yuxiu Mao Weichao 王官锋 The Key Laboratory of Plant Cell Engineering and Germplasm Innovation Ministry of EducationCollege of Life ScienceShandong University

Programmed cell death(PCD) plays important roles in plant innate *** disease resistance(R) genes encode nucleotide binding leucine-rich-repeat(NLR) proteins that trigger a rapid localized PCD termed the hypersensitive response(HR) upon pathogen *** maize NLR protein Rpl-D21 derives from an intragenic recombination between two NLRs,Rpl-D and Rpl-dp2,and confers an autoactive HR in the absence of pathogen ***(MC),the distant homolog of caspase in the caspase-like domain(CD) cysteine protease superfamily,is widely distributed in different species and plays important roles in *** maize,MC can be divided into two types,type Ⅰ and type Ⅱ.Phylogenetic analysis showed that eight genes encode type Ⅰ(ZmMC 1-8) and three genes encode type Ⅱ(ZmMC9-11).Both types of MC contain two conserved caspase-like catalytic domains,P20(20 kDa) and P10(10 kDa).Two types of MC differ in their N-terminal pro-domains and the length of the linker between P20 and *** our RNA-seq data,we found that several ZmMC genes are differentially expressed in Rp1-D21 mutant compared to wild *** transiently co-expressed ZmMC and Rpl-D21 in *** and found that type Ⅰ ZmMC1 and ZmMC2 suppress Rp1-D21-mediated HR,while type Ⅱ ZmMC9 has no obvious *** suppression function of ZmMC1 and ZmMC2 is independent of the putative catalytic residues and the N-terminal *** results suggest that two types of MC have different function in regulating NLR-mediated *** molecular mechanism will be further explored.

关键词： NLR Rp1-D21 Metacaspase Hypersensitive response

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烟草IP-L(Interaction Protein L)互作蛋白的筛选与表达分析

烟草IP-L(Interaction Protein L)互作蛋白的筛选与表达分析

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中国植物病理学会第十一届全国会员代表大会暨2018年学...

作者：蒲运丹张永至孙现超西南大学植物保护学院

IP-L(Interaction Protein L)是从烟草cDNA文库中筛选出的与ToMV外壳蛋白(coat protein,CP)互作的寄主蛋白,两者共定位于本氏烟细胞表皮质膜。ToMV CP和IP-L相互作用可能影响叶绿体的稳定性导致萎黄。通过酵母双杂交筛选本氏烟文库,获... 详细信息

IP-L(Interaction Protein L)是从烟草cDNA文库中筛选出的与ToMV外壳蛋白(coat protein,CP)互作的寄主蛋白,两者共定位于本氏烟细胞表皮质膜。ToMV CP和IP-L相互作用可能影响叶绿体的稳定性导致萎黄。通过酵母双杂交筛选本氏烟文库,获得一个与IP-L互作的蛋白,为了研究其与IP-L之间的相互作用及烟草花叶病毒(Tobacco mosaicvirus,TMV)侵染下的表达变化。采用双酶切的方法构建PGADT7-TSJT1和PGBKT7-IP-L酵母表达载体以及pCV-cYFP-IP-L和pCV-nYFP-TSJT1双分子荧光互补表达载体。将重组载体质粒PGADT7-TSJT1和PGBKT7-IP-L共转化至AH 109酵母菌株,验证烟草IP-L与候选蛋白TSJT1的相互作用;利用农杆菌EHA105介导法在本氏烟中表达双分子荧光互补表达载体pCV-cYFP-IP-L和pCV-nYFPTSJT1,激光共聚焦显微镜下观察相互作用产生的YFP荧光信号;利用实时荧光定量PCR(RTqPCR)分析本氏烟各个组织中TSJT1的表达量,TMV胁迫下及IP-L沉默的本氏烟TSJT1的表达变化情况;构建亚细胞定位载体pCV-mGFP-TSJT1,pRFP-AtRop10作为质膜marker,激光共聚焦观察融合TSJT1的GFP荧光信号定位情况。结果显示与IP-L互作的蛋白分子量为25.6kDa,其对应ORF全长714bp,NCBI对比显示其与预测的多种茄科植物的茎特异蛋白同源性达到99%,与拟南芥的一个铝诱导蛋白具有73.3%的同源性。通过酵母双杂交系统和双分子荧光互补技术进一步确认IP-L与TSJT1的相互作用;RT-qPCR分析显示,TSJT1在本氏烟根、茎、叶、花中均有表达,在叶中表达量最高;亚细胞定位显示TSJT1定位于本氏烟细胞质膜;采用摩擦接种方法,用绿色荧光蛋白(Green Fluorescence Protein,GFP)标记的TMV侵染本氏烟,在接种2d、3d、4d后,TSJT1在叶片内表达量随接种时间呈明显上调趋势。TMV侵染4d后的叶片内表达量是对照组的2倍,并且差异达到显著水平。在TRV介导的IP-L沉默本氏烟中,TSJT1的表达也显著下降。该结果表明,TSJT1与IP-L在本氏烟中有类似的组织表达模式,在叶中表达量最高,且在TMV侵染本氏烟时表达量显著上调。

关键词：本氏烟 TMV Interaction Protein L 互作蛋白

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