Potato root diffusate-induced secretion of soluble, basic proteins originating from the subventral esophageal glands of potato cyst nematodes

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.     

草莓胶孢炭疽菌CFEM候选效应子的生物信息学鉴定及其侵染过程中的转录分析

病原真菌通常分泌效应子到寄主组织中调控寄主的生理过程,从而有利于其侵染。CFEM(common in several fungal extracellular membrane)蛋白是真菌所独有的,且与致病性密切相关。本研究利用Pfam数据库对草莓胶孢炭疽菌全基因组进行搜索,鉴定获得22个CFEM蛋白。对CFEM蛋白的信号肽、跨膜结构域和亚细胞定位进行分析,结果表明仅有8个CFEM蛋白为分泌蛋白。对CFEM分泌蛋白在不同侵染阶段的转录情况进行转录组学及RT-PCR分析,结果显示8个CFEM蛋白在侵染后不同时期均有表达。其中,1个CFEM分泌蛋白于附着胞形成期特异表达,2个于活体寄生阶段特异表达,2个于死体寄生阶段特异表达。综合上述分析结果,预测这8个分泌蛋白可能为草莓胶孢炭疽菌的效应子。本研究为深入解析植物病原真菌CFEM效应子提供了理论依据。     

重要植物寄生线虫内切葡聚糖酶基因研究进展

 在线虫与植物互作的过程中,降解寄主细胞壁是植物线虫成功建立寄生关系的关键环节。β-1,4-内切葡聚糖酶(β-1,4-endoglucanase,ENG)是由线虫食道腺细胞产生并由口针分泌、对细胞壁降解起关键作用的酶类之一。本文对近年来植物寄生线虫eng基因的克隆、组织定位、表达分析、基因、编码蛋白的结构功能以及ENG来源、进化及在线虫与植物互作中的潜在作用等进行了概述。     

Immunodetection of elicitins from Phytophthora spp. using monoclonal antibodies

Ten monoclonal antibodies were selected from mice immunized with a highly purified elicitin secreted by Phytophthora cryptogea , termed cryptogein. These antibodies could be classified into five groups according to their cross-reactivity to heterologous elicitins from other Phytophthora species, from strict specificity (reacting solely with cryptogein) to broad reactivity (reacting with all four elicitins under study). When examined on BIA core (a real-time biospecific interaction analyser), these monoclonal antibodies were found to recognize at least three different epitopes on the cryptogein molecule. Their use in elicitin detection and quantification was optimized in several ELISA protocols. A mixed monoclonal-polyclonal antibody, indirect DAS-ELISA procedure detected as little as 20 pg of purified elicitin per well (100 μl). The four elicitins could be detected with the aid of one of couple of polyvalent reagents, whilst each one could be detected separately using appropriate monoclonal antibodies. These protocols have been used to detect elicitins secreted by Phytophthora spp. into culture medium as well as in planta following plant inoculation.     

高温处理西花蓟马若虫对其雌成虫寿命、繁殖力及后代发育的影响

温度是影响昆虫生长发育的重要生态因素之一,高温逆境作为一种绿色防控手段可以有效控制温室害虫种群发展。为了探究高温处理西花蓟马若虫对其种群发展的影响,本试验用41℃和43℃两个温度分别处理西花蓟马初孵若虫2、6、12、24和36h后,观察并记录其雌成虫寿命、繁殖力及后代发育指标的变化情况。结果表明,随着处理时间的延长,两个高温处理后西花蓟马雌成虫寿命,子代若虫总数、成虫总数和总存活率(若虫发育到成虫的存活率)均显著下降,后代雌雄性比呈总体下降趋势。41℃处理后,性比从2.30∶1降低到2.13∶1;43℃处理后从2.25∶1降低到2.07∶1,均明显低于对照的2.69∶1。另外,两性生殖种群相比于孤雌生殖种群更易遭受高温处理的影响。     

The secreted proteome of necrotrophic Ciborinia camelliae causes nonhost-specific virulence

Ciborinia camelliae (Sclerotiniaceae) is a host- and organ-specific fungal pathogen that causes rapid browning and flower drop on ornamental plants of the genus Camellia. To determine the nature of its necrotrophic factors, we tested whether proteins secreted by C. camelliae can damage host-plant tissues. Fungal culture filtrate caused necrogenic activity, abolished by heat or protease treatments, thus indicating that the secreted necrogenic agents are probably proteinaceous in nature. Mass spectrometry-based proteomics was used to detect and identify secreted proteins of C. camelliae. Proteins secreted in culture media (in vitro) and in petal apoplast (in planta) had similar functional distributions, and key identified proteins included homologs to known virulence factors of the closely related Sclerotiniaceae fungus Botrytis cinerea, including endopolygalacturonases, cerato-platanin family proteins, and necrosis- and ethylene-inducing peptides. The main class of secreted proteins were carbohydrate-active enzymes, a characteristic signature of necrotrophic plant pathogens. Both fungal culture filtrate and apoplastic washes of infected petals induced necrosis when infiltrated into host and nonhost plants. This suggests that while some of the secreted proteins might contribute to virulence of C. camelliae, and can cause necrosis similar to secreted proteins of broad-host Sclerotiniaceae pathogens, they do not have a role in determining its host specificity.     

Relationship between physiological response of French beans of different age to Meloidogyne incognita and subsequent yield loss

A study was made of the effect of a single generation of the root-knot nematode Meloidogyne incognita on the growth of potted French bean plants ( Phaseolus vulgaris ) inoculated at different stages of plant maturity. In separate experiments. 3-, 11- and 13-day-old plants were inoculated before primary leaf expansion (BPLE). at the appearance of trifoliate leaves (TRIF) and at the flower bud (BDS) stages respectively, with 0, 2000, 4000 or 8000 second-stage juvenile nematodes and maintained in a growth chamber under controlled conditions. The photosynthetic rate of the plants inoculated at the TRIF and BDS stages decreased significantly with increasing inoculum level 7 days after inoculation. Although the respiration rate did not significantly change throughout the experimental period, the ratio of photosynthetic to respiration rate decreased significantly with increasing nematode inoculum level and duration of infection. Chlorophyll content, plant dry weight and the numbers of buds, flowers, pods and seeds were significantly lower in infected plants than in the controls; this effect increased with increasing levels of nematode inoculum for all three plant stages. The leaf area was significantly smaller only when nematode infection occurred at the BPLE stage. The plants which were youngest at the time of nematode infection produced the lowest yield; this appeared to result from the effect of nematodes on photosynthesis and related physiological processes.     

植物寄生线虫分支酸变位酶基因的研究进展

 植物寄生线虫食道腺中表达的寄生基因编码的分泌蛋白在线虫侵入寄主植物、建立取食位点和抑制寄主的防御反应过程中起重要作用。利用植物寄生线虫食道腺削减cDNA文库及基于同源克隆等方法,鉴定了这些过程中起作用的分支酸变位酶(CM)基因。带有氨基酸末端信号肽的根结线虫CM、孢囊线虫CM与细菌CM的蛋白质序列非常相似。mRNA原位杂交表明,CM基因专门存在于植物寄生线虫的亚腹食道腺中。RT-PCR分析表明,它们的转录丰度在线虫寄生的早期丰度较高,在后期较低或者难以检测到。Southern杂交表明,这些CM基因为多基因家族。CM的蛋白质在专性内寄生线虫中广泛存在,表明这种多功能的酶在控制线虫侵染植物的过程中起重要作用。     

Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Identification of hydrolytic activities expressed by Aspergillus flavus grown on cotton carpel tissue

To identify proteins important for invasion into host plant tissue, Aspergillus flavus was cultured on medium containing cotton carpel tissue as the sole carbon source. We identified several hydrolases suggesting they are important as A. flavus virulence factors for plant colonization. Specifically, Aspergillus flavus AF13 secreted at least two endoxylanase activities and a pectolytic activity when grown on the cotton carpel tissue medium. A concentrated sample derived from the A. flavus growth medium (6-day) was subjected to gel filtration chromatography on a BioGel P-30 column. A major endoxylanase activity was separated from the other fungal-secreted proteins. Additional fungal secreted proteins were partially resolved by gel filtration chromatography on a BioGel P-60 column. Multiple proteins with molecular weights in the 20 to 70 kD range were present in the harvested fungal growth medium. Analysis of these fungal-secreted proteins by liquid chromatography/tandem mass spectrometry identified both endo- and exo-glucanase proteins, α-l-arabinofuranosidase, glucoamylase, α-amylase A, pectate lyase A, xylanase F1, acetylxylan esterase, glutaminase A, as well as conserved hypothetical proteins of unknown function. These proteins likely assist A. flavus in the maceration of plant cell walls, allowing for pathogenic entry and accession of host nutrient resources. Pectolytic and xylanolytic hydrolases, as well as glucanases, appear to be important A. flavus virulence factors.     

Cytological Characterization of Elicitin-Induced Protection in Tobacco Plants Infected by Phytophthora parasitica or Phytoplasma

ABSTRACT Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.     

利用转座子Tn5诱变植物青枯菌获得胞外蛋白输出功能丧失突变体

 通过Tn5诱变技术,分离了植物青枯菌生理小种1号、3号的胞外蛋白输出功能丧失突变体。由于Tn5在其基因组单一的eep位点的插入,突变体失去了向培养滤液分泌产生胞外酶和胞外蛋白质的能力。用碱性磷酸酯酶基因PhoA作为报导基因,研究这些胞外蛋白穿越突变体细胞内膜,结果表明,这些胞外蛋白质可以穿过其内膜,但失去了穿越其外膜的能力。胞外多糖在植物体内和体外的产生没有受到eep基因位点突变的影响。该突变体失去了对番茄植株的致萎能力。     

Expression of the Arabidopsis MCM Gene PROLIFERA During Root-Knot and Cyst Nematode Infection

ABSTRACT Expression of the Arabidopsis thaliana gene PROLIFERA (PRL) was examined during development of root-knot and cyst nematode feeding sites. These obligate plant parasites establish specialized feeding structures in roots that allow them to withdraw nutrients from the host. In the process of establishing feeding sites, nematodes alter cell cycle regulation. PRL is normally expressed specifically in dividing cells at all stages of plant development and was used here as a marker for cell division. PRL expression, reported from a PRL::GUS fusion protein, was detected in nematode feeding sites of both root-knot and cyst nematodes from the earliest stages of infection in both giant cells and syncytia. However, unlike other cell cycle genes, expression of PRL was detected only occasionally in cells surrounding the feeding sites. PRL::GUS activity persisted until late in the infection cycle, past the time when other cell cycle genes are expressed. These data indicate that some aspects of the PRL expression pattern during nematode infection differ from that of other cell cycle genes.     

植物寄生线虫效应蛋白调控寄主防卫反应分子机制研究进展

 在植物寄生线虫与寄主互作过程中,线虫分泌器官如食道腺细胞分泌的效应蛋白在寄主细胞壁修饰和调控寄主免疫反应以及取食位点形成和维护中发挥着关键作用。解析植物寄生线虫关键效应蛋白的功能及其与寄主互作机制将为探索植物寄生线虫防控新策略提供重要的理论基础。本文从效应蛋白降解寄主细胞壁、调控寄主基础免疫反应、诱导免疫反应机制和介导翻译后修饰调控寄主免疫反应以及植物激素代谢途径的调控机制等方面进行了概述。     

植物寄生线虫效应蛋白调控寄主防卫反应分子机制研究进展

 在植物寄生线虫与寄主互作过程中,线虫分泌器官如食道腺细胞分泌的效应蛋白在寄主细胞壁修饰和调控寄主免疫反应以及取食位点形成和维护中发挥着关键作用。解析植物寄生线虫关键效应蛋白的功能及其与寄主互作机制将为探索植物寄生线虫防控新策略提供重要的理论基础。本文从效应蛋白降解寄主细胞壁、调控寄主基础免疫反应、诱导免疫反应机制和介导翻译后修饰调控寄主免疫反应以及植物激素代谢途径的调控机制等方面进行了概述。     

Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens

Chitinase modifying proteins, cmps, are secreted fungal proteases that truncate specific plant class IV chitinases by cleaving peptide bonds in their amino termini. We recently identified a cmp from the Zea mays (maize) pathogen Fusarium verticillioides and found that it is a member of the fungalysin class of proteases. We also found that Alternaria brassicae, a pathogen of the mustard plant family Brassicaceae, secretes a protease with the same activity. To determine which pathogens of Brassicaceae plants secrete fungalysin cmps, we tested protein extracts from twenty fungi that had been isolated from diseased plants. Each fungal isolate was grown saprotrophically on maize and canola seeds. Secreted fungal proteins were extracted from cultures and incubated with three purified plant chitinases: ChitA and ChitB from maize, and AtchitIV3 from Arabidopsis thaliana. We found that fungalysin cmps were secreted by fungal pathogens distributed among five families in three major Ascomycota classes (Dothideomycetes, Leotiomycetes and Sordariomycetes). Of four fungal species that did not secrete fungalysin cmp activity, three secreted other cmps that truncated maize ChitA and ChitB by cleaving other peptide bonds. AtchitIV3 was only susceptible to truncation by fungalysin cmps. These results show that cmps are commonly secreted by fungal pathogens of Brassicaceae and suggest that interfering with fungalysin cmp activity may improve plant resistance to multiple fungal diseases.     

Local and Systemic Induced Resistance to the Root-Knot Nematode in Tomato by DL-beta-Amino-n-Butyric Acid

ABSTRACT Chemical inducers of pathogenesis-related proteins and plant resistance were applied to tomato plants, with the aim of inducing resistance to the root-knot nematode Meloidogyne javanica. Relative to control plants, foliar spray and soil-drenching with dl-beta-amino-n-butyric acid (BABA) reduced root-galling 7 days after inoculation, as well as the number of eggs 30 days after inoculation. Other chemicals (alpha- and gamma-amino-n-butyric acid, jasmonic acid, methyl jasmonate, and salicylic acid) were either phytotoxic to tomato plants or did not improve control of root-knot nematodes. Fewer second-stage juveniles invaded BABA-treated tomato roots, and root-galling indices were lower than in control tomato plants. Resistance phenomena in seedlings lasted at least 5 days after spraying with BABA. Nematodes invading the roots of BABA-treated seedlings induced small, vacuolate giant cells. Postinfection treatment of tomato plants with BABA inhibited nematode development. It is speculated that after BABA application tomato roots become less attractive to root-knot nematodes, physically harder to invade, or some substance(s) inhibiting nematode or nematode feeding-site development is produced in roots.     

Transient expression of gfp in the obligate biotrophic oomycete Plasmopara halstedii using electroporation and a mechanoperforation method

Studying plant biotrophic oomycetes is difficult, because they depend on living plant cells for nutrition, which necessitates investigations in planta . Internal optical labelling is a powerful tool for analysing intra- and interspecific interactions. Different approaches are described for establishing a gfp -transformation system for Plasmopara halstedii , the causal agent of sunflower downy mildew. A vector containing gfp and the oomycete-specific ham34 regulators from Bremia lactucae was constructed for transformation experiments. Particle bombardment of infected sunflower cotyledons during different developmental stages of the pathogen did not result in successful transformation. In contrast, transient gfp expression in sporangia was achieved using electroporation. However, gfp expression was lost during the subsequent round of infection. A novel transformation method for biotrophic organisms in planta employing mechanoperforation – so-called Löchern–resulted in sporangiophores of P. halstedii transiently expressing gfp and emerging distant from the site of transformation. The new technique is advantageous compared with others as the transformed hyphae can recover during their vegetative growth, before reproductive structures occur. This first step lays an important foundation for further investigations of obligate biotrophic organisms.     

A biotechnological strategy involving monoclonal antibodies for improvement of potato farming by identification and quantification of potato cyst nematodes in soil samples1

The two closely related nematode species Globodera rostochiensis and G. pallida are one of the major problems encountered in potato cultivation. There is a spectrum of potato plant genes known, which confer resistance to these species and their pathotypes. Potato growing in The Netherlands has to follow strict rules to control spread of the pests. Since distinction between the two nematode species is difficult, a rapid and reliable identification method is needed to allow better use of existing and forthcoming resistant potato cultivars. The aims of this project were: (1) identification and partial purification of species-specific proteins from the nematodes, (2) production of species-specific monoclonal antibodies, and (3) development of a screening test for qualitative and quantitative determination of Globodera spp. in soil samples.