A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes

The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules. Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling. The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development. The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity. From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin). Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development. This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.     

A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses

Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria (rhizobia) and mycorrhizal fungi. Nodulation by rhizobia results from a signal transduction pathway induced in legume roots by rhizobial Nod factors. DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs). This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.     

Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.     

华癸中生根瘤菌(Mesorhizobium huakuii)研究进展

综述了华癸中生根瘤菌(Mesorhizobium huakuii)遗传多样性、质粒多样性、质粒的功能、结瘤因子、结瘤基因、胞外多糖等方面的研究进展。这类菌只能在紫云英上结瘤,1997年并入Mesorhizobium。选取大量菌株进行REP-PCR、16S和23S rDNAPCR-RFLP等分析,揭示其分为16种16S rDNA基因型。选用不同16S rDNA基因型的部分代表菌株进行部分16S rDNA测序。能够在紫云英上结瘤的根瘤菌可以分为2个类群。表明这类菌具有遗传多样性,存在不同的种。它们多数含共生质粒,共生质粒定位于最大或次大质粒上,质粒数1~5个不等,其分子量范围在53~906 kb之间。质粒缺失菌株表现为不结瘤(Nod-)和结瘤而不固氮(Nod ,Nif-)。某些质粒缺失还影响LPS合成、抗酸性、竞争结瘤能力。其结瘤因子能诱导紫云英根毛变形,它们的结瘤因子结构类似于苜蓿根瘤菌的结瘤因子。它们的结瘤基因具独特性,nodA与nodBC分隔一定距离,且具有保守性。它们产生的胞外多糖在侵染结瘤过程中发挥重要作用。     

LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia

Rhizobium-root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor-like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis.     

豆科植物根瘤茵结瘤因子的感知与信号转导

结瘤因子(脂壳寡糖,lipo-chito-oligosaccharides,LCOs)是根瘤菌在宿主植物根系分泌的类黄酮的作用下,合成并分泌的一类多糖信号分子,在根瘤菌与植物的共生结瘤过程中起重要作用。结瘤因子通过一定的机制感知,与某种特定受体(结瘤因子结合蛋白)结合,通过结瘤因子激活的信号转导途径如Ca2 介导的信号转导途径或磷酸类脂信号转导途径,诱导宿主植物的一系列反应,如根毛质膜去极化、皮层细胞分裂和结瘤素基因表达等。同时,结瘤因子的感知与信号转导也受一定基因和反馈机制调控。就豆科植物根瘤菌结瘤因子感知机制、信号转导途径及反馈调控等方面的研究进展进行了全面阐述。     

LysM domain receptor kinases regulating rhizobial Nod factor-induced infection

The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor. We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection. The SYM2 orthologous region of M. truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs). Using reverse genetics in M. truncatula, we show that two LYK genes are specifically involved in infection thread formation. This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.     

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.     

Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract

The gene encoding the Nod2 protein is frequently mutated in Crohn's disease (CD) patients, although the physiological function of Nod2 in the intestine remains elusive. Here we show that protective immunity mediated by Nod2 recognition of bacterial muramyl dipeptide is abolished in Nod2-deficient mice. These animals are susceptible to bacterial infection via the oral route but not through intravenous or peritoneal delivery. Nod2 is required for the expression of a subgroup of intestinal anti-microbial peptides, known as cryptdins. The Nod2 protein is thus a critical regulator of bacterial immunity within the intestine, providing a possible mechanism for Nod2 mutations in CD.     

转地蜂群病原微生物及肠道共生菌的变化

【目的】探明转地放蜂过程中常见蜜蜂病毒和寄生虫的流行规律,及不同地区工蜂肠道中两种主要共生菌Gilliamella apicola和Snodgrassella alvi的变化情况。【方法】在转地放蜂过程中,对同一意大利蜜蜂(Apis mellifera ligustica)蜂场的固定蜂群连续取样,采用RT-PCR方法检测蜂群中病毒和寄生虫的感染情况,使用SPSS 17.0软件对不同地区、不同季节样本的病毒和寄生虫感染率进行卡方检验。以蜜蜂β-actin为内参基因,对不同地区样本中的共生菌G.apicola和S.alvi进行荧光定量PCR分析,检测转地过程中工蜂肠道中这两种细菌的变化情况,并采用Kendall Rank相关系数对病原物感染率和共生菌含量进行相关性分析。【结果】转地放蜂7个地区的样本中,仅检测出以色列急性麻痹病毒(IAPV)、黑蜂王台病毒(BQCV)和蜜蜂残翅病毒(DWV)3种蜜蜂病毒。其中IAPV和BQCV在所有地区均有检出且感染率较高,不同地区之间感染率差异显著;DWV感染率相对较低,不同地区之间感染率差异极显著。西方蜜蜂微孢子虫(Nosema apis)在各地区样本中均未检出,东方蜜蜂微孢子虫(Nosema ceranae)在4个地区的样本中检出,且不同地区间感染率差异极显著,熊蜂微孢子虫(Nosema bombi)在各个地区均有检出,不同地区间感染率差异显著;季节性差异分析表明,IAPV在不同季节的感染率差异不显著,而BQCV、DWV、N.ceranae和N.bombi在不同季节的感染率差异显著,且春夏季的感染率普遍高于秋冬季;不同转地地区的工蜂肠道内均含有共生菌G.apicola和S.alvi,且两种共生菌含量在不同地区间均差异极显著;相关性分析表明,S.alvi和IAPV之间呈显著负相关作用。【结论】转地蜂场工蜂病原微生物的检测结果表明IAPV、BQCV、DWV和微孢子虫在蜂群中普遍存在;蜜蜂病原物的感染率和肠道共生菌的含量在不同地理区域间差异显著;部分病原物与肠道共生菌间呈显著负相关关系;转地放蜂方式对蜜蜂的健康状况有一定影响。     

Nutritional significance of symbiotic bacteria in two species of hemoflagellates

Symbiote-free strains of Blastocrithidia culicis and Crithidia oncopelti, obtained by chloramphenicol treatment, were compared nutritionally with normal, symbiote-containing strains. The symbiotic bacteria spare the flagellates requirements for exogenous hemin and for other nutritional factors present in liver extract.     

Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium

Root nodules of leguminous plants are symbiotic organs in which Rhizobium bacteria fix nitrogen. Their formation requires the induction of a nodule meristem and the formation of a tubular structure, the infection thread, through which the rhizobia reach the nodule primordium. In the Rhizobium host plants pea and vetch, pre-infection thread structures always preceded the formation of infection threads. These structures consisted of cytoplasmic bridges traversing the central vacuole of outer cortical root cells, aligned in radial rows. In vetch, the site of the infection thread was determined by the plant rather than by the invading rhizobia. Like nodule primordia, pre-infection thread structures could be induced in the absence of rhizobia provided that mitogenic lipo-oligosaccharides produced by Rhizobium leguminosarum biovar viciae were added to the plant. In this case, cells in the two outer cortical cell layers containing cytoplasmic bridges may have formed root hairs. A common morphogenetic pathway may be shared in the formation of root hairs and infection threads.     

Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors

Although much is known about the bacterial genetics of early nodulation, little is known about the plant cell response. Alfalfa root hair cells were impaled with intracellular microelectrodes to measure a membrane potential depolarizing activity in Rhizobium meliloti cell-free filtrates, a plant response dependent on the bacterial nodulation genes. The depolarization was desensitized by repeated exposure to factors and was not observed in a representative nonlegume. A purified extracellular Nod factor, NodRm-IV(S), caused membrane potential depolarization at nanomolar concentrations. This rapid single-cell assay provides a tool for dissecting the mechanisms of host cell response in early nodulation.     

昆虫病原线虫共生菌对玉米弯孢菌的抑菌活性及其抗逆性

利用从国内采集土样中收集到的23个品系昆虫病原线虫和5个本室保存的线虫品系分离共生菌,通过对共生菌菌株发酵液和初提物抑菌活性的测定,得到1株对玉米弯孢菌具有良好抑菌活性的共生菌菌株SY5,抑菌圈平均直径分别为50.00mm和33.34mm,并对抑菌活性强的菌株发酵液的抗逆性进行了初步探索,结果表明,共生菌菌株SY5在50℃下处理60 min和100℃高温下10 min,其抑菌活性依旧很高;18W紫外线照射120 min对共生菌SY5的抑菌活性的影响不明显;常温保存150d,抑菌活性下降。     

2株昆虫病原线虫共生菌的分离与初步分类鉴定

从陕西杨凌采集的昆虫病原线虫S teinernem a sp.YL 001和S teinernem a sp.YL 002肠道内分别分离到1株具有较高杀虫和抑菌活性的共生菌菌株YL 001和YL 002,并对其从形态特征、培养特性及生理生化特征等方面进行了鉴定。结果表明,YL 001和YL 002菌株分别为嗜线虫致病杆菌(X enorhabdus nem a toph ila)和伯氏致病杆菌(X enorhabdus bov ien ii)。     

黑曲霉拮抗菌Xenorhabdus bovienii445筛选、鉴定及发酵优化

【目的】从现有共生细菌资源中筛选对黑曲霉具有高拮抗作用的菌株,为寻求安全高效的生物保鲜材料提供更多选择。【方法】采用平板对峙法和琼脂扩散法,分别对现有昆虫病原线虫共生菌进行初筛和复筛,对筛选出的高效拮抗共生细菌进行生理生化以及16S rRNA 序列进化分析鉴定,通过单因素试验和响应面分析法优化菌株培养条件,利用损伤接种法在红地球葡萄上验证对黑曲霉防治效果。【结果】分离筛选共获得20株拮抗共生菌,复筛得到1株抑菌效果显著的共生细菌(445),经生理生化及分子生物学分析为伯氏致病杆菌Xenorhabdus bovienii,GeneBank ID:OK560680,与菌株Xenorhabdus bovienii strain Xb139 (MG995576.1)聚于同一分支,相似性达99.79%。获得445菌株抑菌活性的最优培养条件为接种量3.06 %、pH 7.0、装液量100.15 mL/250 mL,抑菌圈直径为(29.67±0.28) mm,抑菌效价为10.59 cm/mL,比优化前提高39.16%。Xenorhabdus bovienii 445发酵液对黑曲霉具有较好防治效果,3 d后防效为63.50%。【结论】筛选得到1株高效拮抗黑曲霉的昆虫病原线虫共生细菌,其发酵液对黑曲霉有良好的抑制效果,具有较好的生防潜能。