Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying Erwinia psidii pathogenicity

Erwinia psidii (Eps) is the causal agent of emerging diseases of eucalypt and guava; however, the mechanisms underlying its pathogenicity are not fully understood. Here, we predicted factors involved in the ability of Eps to cause disease on its host plants. For that, the genomes of four Eps strains exhibiting different virulence on eucalypt were sequenced, and hrp/hrc genes coding for the type III secretion system (T3SS), effectors injected into the plant cell cytoplasm through the T3SS (T3SEs) and their plant subcellular localizations, as well as proteins deployed to the host apoplast, were predicted. It was found that Eps possesses a complete hrp/hrc gene cluster based on comparison with Erwinia amylovora. A total of 18 T3SEs were predicted, 11 of which were shared among all strains, none were exclusive to any strain and seven were absent in at least one strain. No sequence variation among strains was found for five T3SE candidates whereas extensive variation was found for six, suggesting the latter may be determinants of virulence differences. The T3SE candidates are predicted to target the plant cell nucleus, cytoplasm, mitochondrion, chloroplast and peroxisome. The predicted apoplastic effector repertoire common to all four strains was over-represented in proteins of unknown functions or predicted to possess enzymatic activities, among which the most abundant were oxidoreductases and peptidases. Proteins with lytic transglycosylase activity were predicted in strain-specific apoplastic effector repertoires. These results provide an important framework for future research aimed at uncovering the factors underlying Eps pathogenicity.     

水稻白叶枯病菌Zur和HrpG平行调控hrcC基因表达

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)的III型分泌系统(Type III secretion system, T3SS)由hrp基因簇编码,其决定在寄主水稻上的致病性。hrcC是hrp基因簇中hrpA转录单元仅有的一个基因,推测编码T3SS的核心组分蛋白。在Xoo中,hrcC在致病性中的功能以及受调控的机制仍未明确。本研究构建了hrcC的缺失突变体及其功能互补子,发现hrcC缺失使Xoo丧失了在寄主水稻上的致病性以及在非寄主烟草上激发过敏反应(Hypersensitive response, HR)的能力,功能互补子能够恢复这些表型至野生型水平。启动子GUS活性的定量测定和蛋白免疫杂交试验,证明hrcC的转录表达依赖于主要的hrp调控子HrpG,而不受HrpX调控;HrpG和铁转运家族类调控子(Ferric uptake regulator family)Zur以平行独立的方式正调控hrcC基因的转录表达。异源功能互补、启动子活性和蛋白表达试验发现Xoo和水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)的hrcC基因在致病性上的功能具有互置性,以及受HrpG、HrpX和Zur的调控模式也具有相似性。这些研究为进一步解析黄单胞菌的hrp调控网络与全毒性调控网络之间的交叉提供了新的线索。     

迟钝爱德华氏菌Ⅲ型分泌系统基因的克隆及多重PCR检测方法的建立

采用PCR法扩增大菱鲆Scophthalmus maximus出血性败血症病原菌迟钝爱德华氏菌Edwardsiella tarda L-49231菌株Ⅲ型分泌系统(TTSS)的装置蛋白(esaC)、效应蛋白(eseB、eseC、eseD)和调节蛋白(esrA、esrB)6种基因,并将其克隆到载体PMD19-T中分别进行测序;通过优化反应条件,建立一次检出效应蛋白eseB、eseC和eseD 3种基因片段的多重PCR方法;采用所建立的多重PCR方法检测迟钝爱德华氏菌不同菌株(L-49231、GhAn080310、GhAn080313和GhAn080314)、沙门氏菌Salmonella enterica、大肠杆菌Escherichia coli和肠杆菌Enterobacter中效应蛋白基因的存在情况.结果表明:L-49231菌株中存在esaC、eseB、eseC、eseD、esrA和esrB 6种基因片段,大小分别为837、184、934、445、464、458 bp,与GenBank 中迟钝爱德华氏菌PPD130/91菌株的这6种基因的同源性分别为99％、97％、99％、99％、99％、98％;通过优化反应条件,在退火温度为54 ℃,基因eseB、eseC和eseD引物浓度(μmol/L)组合比为5∶10∶2,Mg2+浓度为1.5 mmol/L时,能够得到清晰、稳定且均一的多重PCR产物,并与目的条带分子量一致;采用所建立的多重PCR方法进行检测,迟钝爱德华氏菌L-49231菌株中存在eseB、eseC和eseD 3种基因片段,迟钝爱德华氏菌GhAn080310、GhAn080313、GhAn080314菌株中存在eseB和eseC基因片段,但未检出eseD基因片段;沙门氏菌、大肠杆菌和肠杆菌中均未检出eseB、eseC和eseD基因.表明所建立的多重PCR方法对迟钝爱德华氏菌具有较好的特异性,可检出具有Ⅲ型分泌系统效应蛋白的菌株.     

Regulation of pancreatic exocrine secretion in goats: differential effects of short‐ and long‐term duodenal phenylalanine treatment

Four yearling goats (31.2 ± 2.5 kg), surgically fitted with common bile duct reentrant and duodenal catheter, were used in two 4 × 4 Latin square design experiments to investigate the effects of duodenal infusion of phenylalanine for different times on pancreatic exocrine secretion (PES). In experiment 1 (the long‐term experiment), goats were duodenally infused with 0, 2, 4 or 8 g/day phenylalanine for 14 day. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from day 11 to day 14 to encompass a 24‐h day. In experiment 2 (the short‐term experiment), goats were infused with phenylalanine for 10 h continuously at the same infusion rate as experiment 1 after feed deprivation for 24 h repeated every 10 day. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The volume and pH of pancreatic juice were measured, and a 5% subsample was composited and frozen until analysis of enzyme activities. Plasma was frozen until analysis of insulin and cholecystokinin (CCK). In experiment 1, pancreatic juice, α‐amylase secretion and plasma CCK concentration responded quadratically (p < 0.05), with the top value observed at the 2 g/day phenylalanine. Trypsin secretion had a quadratic response (p < 0.05), with secretion increasing up to 4 g/day phenylalanine and decreasing thereafter. Phenylalanine linearly decreased pancreatic protein and lipase secretion (p < 0.05). The results of correlation analysis showed significant correlations (p < 0.05) between plasma CCK concentration and secretion of α‐amylase and trypsin. However, the short‐term phenylalanine infusion did not influence (p > 0.05) pancreatic juice, protein, α‐amylase, lipase, trypsin secretion and plasma CCK concentration. These results indicate PES of ruminants is stimulated by phenylalanine and is potentially mediated by CCK in the long‐term duodenal infusion treatment, but is not influenced by phenylalanine in the short‐term duodenal infusion treatment.     

棉花与不同作物同穴互作育苗对土壤微生物、 酶活性和根系分泌物的影响

为了探明两苗互作育苗提高棉苗素质的机理,本试验在塑料温棚2030℃、 自然光照条件下,采用532280 mm2具有200方形孔的塑料育苗盘,用土壤作基质,以棉花单作育苗为对照,研究了棉花分别与小麦、 玉米、 谷子和高粱同穴互作育苗对土壤微生物、 酶活性及根系分泌物的影响。结果表明, 随互作苗的加入,育苗土壤中细菌和放线菌数量显著增加,真菌数显著降低; 脱氢酶、 中性磷酸酶、 转化酶和脲酶活性显著提高; 2,4-二叔丁基苯酚、 2,6-二叔丁基苯酚、 邻苯二甲酸二丁酯和9-16碳烯酸甲酯根系分泌物的积累量显著减少,棉苗则表现为根系活力、 叶片可溶性糖含量和ATP含量显著提高,增加了棉苗根重、 苗重和侧根数,棉苗离床存活期延长,栽后缓苗期缩短。在四种互作处理中,以棉花+小麦、 棉花+谷子处理中棉苗素质表现较好。综上,两苗同穴互作育苗改善了育苗土壤微生物数量和结构,这可能是提高土壤酶活性和降低土壤有害根系分泌物积累的主要原因,进而提高了棉苗素质。     

光肩星天牛雌虫产卵分泌物与生殖器官内容物组分的比较研究

为了对光肩星天牛(Anoplophora glabripennis)雌虫产卵分泌物与生殖系统内各生殖器官内容物鲜重及组分进行分析比较。通过室内饲养产卵期光肩星天牛成虫采集其产卵分泌物及其生殖系统内生殖器官内容物,利用Bradford法、蒽酮比色法、茚三酮试剂显色法对其组分中蛋白质、可溶性糖、游离氨基酸的含量分析测定。结果表明：产卵分泌物鲜重与卵巢内容物鲜重差异显著,而与交配囊、受精囊腺体及侧输卵管萼内容物鲜重差异不显著。卵巢和交配囊内容物中蛋白质、可溶性糖及游离氨基酸的含量均显著高于产卵分泌物中蛋白质和可溶性糖的含量;侧输卵管萼内容物中蛋白质含量显著低于产卵分泌物中蛋白质含量、可溶性糖及游离氨基酸含量均与产卵分泌物中含量差异不明显;受精囊腺内容物中蛋白质、可溶性糖及游离氨基酸含量与产卵分泌物中含量差异不显著,其组分与产卵分泌物中组分基本一致。光肩星天牛雌虫受精囊腺为其产卵分泌物的贮藏器官。     

苏云金芽胞杆菌CT-43内生质粒pBMB0558上virD4基因的功能

苏云金芽胞杆菌CT-43高产苏云金素,其内生质粒pBMB0558上virD4基因编码的VirD4蛋白与Ⅳ型分泌系统中结合底物的偶合蛋白(T4CPs)高度同源.本研究利用插入缺失突变方法,构建同源双交换载体,敲除CT-43中的virD4基因,得到1株突变株BMB1122.HPLC检测结果显示,突变株BMB1122发酵上清...     

木香薷叶表皮毛的类型、分布及腺毛的分泌过程

采用光学显微镜、扫描电镜及Neu’s试剂染色法结合荧光显微观察,研究了木香薷叶表皮毛的类型、分布及腺毛分泌过程中外部形态的变化和分泌黄酮类物质的过程。结果显示:(1)木香薷叶表皮有腺毛和非腺毛。腺毛包括盾状腺毛和头状腺毛。成熟的盾状腺毛头部较大,呈盾状,直径为(68.14±0.84)μm。成熟的头状腺毛头部较小,呈半圆球形,直径为(17.57±0.48)μm。非腺毛包括乳突状毛、分枝状毛和管状毛。(2)叶片远轴面和近轴面均有表皮毛分布。腺毛在远轴面分布较多,近轴面较少;盾状腺毛主要分布在远轴面,头状腺毛则两面均有分布。非腺毛在叶表皮两面均有分布,以叶脉周围、叶片边缘和基部分布较多。(3)成熟腺毛的分泌物储存于其头部外皮下的间隙,随着分泌物的增加,外皮凹陷或破裂,释放出分泌物,腺毛溃落。(4)盾状腺毛和头状腺毛均能分泌黄酮类物质,随着腺毛的发育成熟,黄酮类物质逐渐积累于腺毛头部,随后释放出来。     

无脉苔草根际优良促生菌鉴定及其作用研究

采用钼蓝比色法(molybdenum blue colorimetry, MBC)、乙炔还原法(acetylene reduction assay, ARA)及高效液相色谱法(high performance liquid chromatography, HPLC)测定了分离自无脉苔草根际的6株根际促生菌溶磷、固氮及分泌激素的特性,并利用16S rDNA序列分析技术对筛选出的2株优良促生菌进行鉴定。结果表明,6株促生菌的溶磷量在298.17~554.67 mg/L之间,其中菌株TPRS3的溶磷能力最强(554.67 mg/L);6株促生菌中有4株具有固氮能力,固氮酶活性在170.19~456.87 nmol C₂H₄/(mL·h)之间,菌株TPRS19的固氮能力最强,固氮酶活性为456.87 nmol C₂H₄/(mL·h),菌株TRPS3和TPRS12无固氮能力;6株促生菌均具有分泌植物激素的能力,其中3-吲哚乙酸(IAA)分泌量在8.20~86.36 mg/L范围内,赤霉素(GA₃)在26.36~135.90 mg/L范围内,反-玉米素(t-Z)在9.55~141.68 mg/L范围内,菌株TPRS19分泌IAA的能力最强,为86.36 mg/L,菌株TPRS5分泌GA₃的能力最强,为135.90 mg/L,菌株TPRS2分泌t-Z的能力最强,为141.68 mg/L,菌株TPRS3、TPRS5、TPRS12和TPRS19兼具分泌3种激素能力。经鉴定,菌株TPRS5为枯草芽孢杆菌(Bacillus subtilis),菌株TPRS19为阴沟肠杆菌(Enterobacter cloacae),这2株菌有望作为研制微生物肥料的优良菌株。     

III型分泌系统是欧美杨溃疡病菌Lonsdalea quercina重要的致病因子

 欧美杨溃疡病是由Lonsdalea quercina(原称Brenneria quercina,Erwinia quercina)引起的一种细菌性病害,2005年首次在我国河南发现,对我国杨树产业造成了严重影响。菌株N-5-1是从河南濮阳自然发病杨树枝条上分离到的致病菌株。根据N-5-1菌株全基因组测序的初步结果分析,发现N-5-1具有完整的III型分泌系统(Type III secretion system,T3SS)。该系统与植物病原菌Erwinia amylovora CFBP1430和Dickeya dadantii 3937的T3SS高度相似,共由26个基因编码,共约23 kb,其中9个为保守的hrc基因。将L. quercina N-5-1菌株T3SS中保守的结构基因hrcV进行缺失突变,生物测定发现ΔhrcV突变体对“中林46杨”(Populus ×euramericana ‘Zhonglin 46’)2年生枝条的致病力明显下降,而互补菌株HBhrcV致病力与野生菌株保持一致。表明该菌中T3SS是病原细菌重要的致病性因子。菌株N-5-1中hrcV基因的突变导致诱导烟草过敏性坏死反应能力丧失,但并没有影响菌株的生长速率、游动性和生物膜的形成。本研究首次证明了L. quercina N-5-1菌株的T3SS是重要的致病因子。