Molecular Cloning of PbSTKL1 Gene from Plasmodiophora brassicae Expressed during Clubroot Development

Infection of crucifers by the obligate plant pathogen Plasmodiophora brassicae Woron. results in the formation of clubroot disease in these plants. Plasmodiophora brassicae gene expression during disease development was studied by differential display analysis of total RNA extracted from the roots of Chinese cabbage inoculated with the pathogen. In a series of experiments, 30 differentially expressed bands of cDNA were detected, and the expression of clone no. 17 was confirmed in clubbed roots. Southern blot analysis showed that this clone was a single‐copy gene in the P. brassicae genome. Putative amino acid sequence analysis of the full‐length cDNA of clone no. 17 (4.6 kb, designated PbSTKL1) revealed a serine/threonine kinase‐like domain at the C‐terminal region and a coiled‐coil structure in the middle region of the putative protein. PbSTKL1 expression increased strongly beginning 30 days after inoculation and was coincident with resting spore formation.     

Resistance to attack by Brevicoryne brassicae among plants of Brussels sprouts

Because they remained almost uncolonized by the cabbage aphid (Brevicoryne brassicae (L.)) throughout the growing season, plants of Brussels sprouts were singled out in each of 4 years, from plots heavily infested with the aphid, as possibly being resistant to attack. Clones of these plants were established from cuttings and tested in a controlled environment by inoculation with B. brassicae and later, in the field, by natural infestation. The tests confirmed that some of the plants were resistant to the aphid, and the most resistant of those from the first year of the work proved at least as resistant as any subsequently found. The resistance was expressed as antibiosis, but in the field host non-preference was also shown by incoming winged aphids. The possibility that biotypes of B. brassicae might exist, to which the resistant sprout clones were not necessarily resistant, was investigated using B. brassicae collected from sprouts from each of several areas in England. Eight sprout clones, seven of which were known to be resistant, and the other susceptible, to B. brassicae from Wellesbourne were tested with these other B. brassicae. The results showed that biotypes of the aphid, with differing abilities to colonize respective sprout clones, existed in each area, and of the seven sprout clones resistant to the Wellesbourne aphid, only one appeared never to be fully susceptible to one or more of the other biotypes of B. brassicae.     

Development of a PCR-based Assay for the Detection of Plasmodiophora brassicae in Soil

A single-tube nested polymerase chain reaction (STN PCR) method was developed for detecting the causal agent of clubroot disease, Plasmodiophora brassicae. Outer primer PBTZS-2 (5′-CCGAATTCGCGTCAGCGTGA-3′) to amplify a 1457 bp-fragment from P. brassicae DNA and nested primers, PBTZS-3 (5′-CCACGTCGATCACGTTGCAAT-3′) and PBTZS-4 (5′-GCTGGCGTTGATGTACTGGAA-TT-3′), to amplify a 398 bp-fragment internal of the 1457 bp-fragment were used for the STN PCR. The 398 bp-fragment was amplified from as little as 1 fg of P. brassicae DNA with the STN PCR. A protocol for extracting P. brassicae DNA directly from soil was developed. By using the protocol, DNA was extracted from artificially infested soil containing various numbers of P. brassicae resting spores and the resulting DNA was used as template for the STN PCR. As little as one resting spore of P. brassicae per g of soil was detectable with the STN PCR. The STN PCR was applied to naturally infested soil from 3 fields and one canal bed. The 398 bp-fragment was amplified from soil of 2 fields and the canal bed. To improve the detection of P. brassicae, the STN PCR products were subjected to second PCR amplification (double PCR) using the nested primers PBTZS-3 and PBTZS-4. The double PCR amplification generated a single 398 bp-DNA band which was visualized clearly on the agarose gel for all the 4 soil samples tested. A combination of the STN PCR and the double PCR appears a useful assay method for detecting P. brassicae resting spores in field soil.     

Host range and virulence of five baculoviruses from lepidopterous hosts

The host range and virulence of five insect baculoviruses (two multiply-enveloped nuclear polyhedrosis viruses (MNPVs) from Agrotis segetum and Mamestra brassicae; one singly-enveloped NPV from Plusia gamma and two granulosis viruses (GVs) from A. segetum and Pieris brassicae) were studied for seven lepidopterous pests of temperate agriculture (A. segetum, Agrotis exclamationis, Lacanobia oleracea, M. brassicae, Noctua pronuba, P. gamma and Pieris rapae). None of the viruses killed all species but M. brassicae MNPV failed to infect only P. rapae. The other viruses were restricted to the homologous host, or members of its genus or subfamily. In all examples except A. segetum GV, the median lethal dose for the most susceptible host, was less than 22 virus inclusion bodies and median lethal times for all infections ranged from 5·5 to 16·6 days. The low susceptibility of A. segetum and other noctuids to GV infections is discussed in relation to the structure of inclusion bodies and the nature of the infectious unit in baculoviruses.     

A Detached Cotyledon Test for the Isolation of Mutants of Pyrenopeziza brassicae Defective in Pathogenicity Determinants

A detached cotyledon pathogenicity test was devised for the isolation of UV-induced mutants of the hemibiotrophie ascomycete pathogen of Brassica spp., Pyrenopeziza brassicae defective in pathogenicity determinants. At least 95 % of cotyledons of suitable susceptible cultivars of oilseed rape (Brassica napus L. ssp. oleifera cvs Shogun or Bolko) showed symptoms of disease (white spore pustules [conidiomata] on the surface of the cotyledon) within three weeks of inoculation of cotyledons with 10⁴ conidia of P. brassicae. The test allowed rapid screening of UV-induced mutants with a low frequency of false negatives. From 1,700 survivors of UV mutagenesis tested, 20 non-pathogenic mutants were obtained.     

根肿菌侵染下菘蓝生物碱合成机制

为探究根肿菌胁迫对菘蓝生物碱及其合成关键酶基因表达的影响,该研究对根肿菌侵染后0、7、14、21 d的菘蓝进行病情形态分级、组织学观察、生理生化指标测定以及转录组学和代谢组学分析。结果表明:(1)接菌后0、7、14、21 d菘蓝根部分别发展为0级、1级、3级、5级的肿根,并且7 d是皮层入侵的关键时间点。(2)接种根肿菌14 d后,菘蓝叶内可溶性蛋白、丙二醛的含量,超氧化物歧化酶、过氧化物酶、多酚氧化酶、过氧化氢酶的活性与同时间对照组比较显著提高,并随着接菌时间的延长呈增加的趋势。(3)代谢组学一共检测到161种生物碱,其中吲哚类生物碱数量较多; 与未接菌相比,菘蓝接菌后7、14、21 d分别存在16种、17种、39种差异代谢物且各组差异代谢物多富集在生物碱和氨基酸代谢通路。(4)转录组测序结果显示,与未接菌相比,菘蓝接菌后7、14、21 d分别存在2 439个、256个、6 437个差异表达基因,这3组共同富集到11个生物碱相关的代谢通路; 与未接菌相比,接菌后7、14、21 d有9个基因(编码4种酶THS、TAT、YUCCA、ALDH)表达量均上升。该研究结果揭示了芸薹根肿菌与菘蓝之间的互作机制,探究了芸薹根肿菌对吲哚生物碱合成及其关键酶基因的影响,为后期菘蓝根肿病抗性基因及生物碱次生代谢途径的研究奠定了基础。     

Varietal differences in the susceptibility of Brussels sprouts to lepidopterous pests

The attack by cabbage caterpillars on open-pollinated cultivars of Brussels sprouts at Wellesbourne was recorded approximately weekly from July to October in 1973 and 1974. The red-foliaged cv. Rubine was much less attacked by Pieris rapae than any of the twenty-five green-foliaged cultivars tested, the least attacked of which were The Aristocrat, Continuity, Evergreen and Pride of the Market. Rubine also showed some resistance to attack by Mamestra brassicae and Evergestis forficialis, as did The Aristocrat. Continuity, though as resistant as any of the cultivars to E. forficialis, was not resistant to M. brassicae. No evidence of antibiosis was found and resistance, based on non-preference, is considered to result from ovipositing P. rapae adults discriminating against the red colour of Rubine and against the leaf composition (relative volatile content) of the green-foliaged resistants. This could also apply with M. brassicae and E. forficialis.     

Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA‐Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.     

The role of host species, age and defensive behaviour on ovipositional decisions in a solitary specialist and gregarious generalist parasitoid (Cotesia species)

The main objective of this study was to determine the extent to which host acceptance behaviour as related to host species, age, and defensive behaviour might explain the differences in host use that exist between two congeneric and sympatric species of parasitic wasps. Cotesia glomerata (L.) (Hymenoptera: Braconidae) is gregarious and generalist on several species of Pieridae, whereas C. rubecula (Marshall) is solitary and specific to Pieris rapae (L.). Cotesia species differed in their responses to host species (P. brassicae (L.), P. napi (L.) and P. rapae) and developmental stage (early and late 1st, 2nd and 3rd instars). In no-choice tests, host acceptance by C. rubecula was higher for p. rapae and females did not distinguish among the 6 host ages. In contrast, when foraging for P. brassicae and P. napi, C. rubecula females more readily attacked early first instar. Cotesia glomerata showed a higher degree of behavioural plasticity towards acceptance of Pieris host species and host age than did C. rubecula. Cotesia glomerata females parasitized the three Pieris species and showed higher acceptance of first and second instars over third instar. Oviposition success was also influenced by host defensive behaviour. The frequency and the effectiveness of defensive behaviour rose with increasing age of the host, P. brassicae being the most aggressive Pieris species. Furthermore, the mean duration of C. glomerata oviposition was significantly reduced by the defensive reactions of P. brassicae, which would likely affect parasitoid fitness as oviposition time is positively correlated to clutch size in C. glomerata. Acceptance frequencies corresponded well to field reports of Pieris-Cotesia associations and to patterns of parasitoid larval performance, suggesting that the acceptance phase might be used as a reliable indicator of Cotesia host-specificity.     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Potential regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway is possibly involved in preeclampsia pathogenesis

Preeclampsia (PE), a pregnancy‐specific disorder, is a leading cause of perinatal maternal‐fetal mortality and morbidity. Impaired cell migration and invasion of trophoblastic cells and an imbalanced systemic maternal inflammatory response have been proposed as potential mechanisms of PE pathogenesis. Comparative analysis between PE placentas and normal placentas profiled differentially expressed miRNAs, lncRNAs, and mRNAs, including miR‐19a‐3p (miRNA), PSG10P (lncRNA), and IL1RAP (mRNA). This study was conducted to investigate their potential roles in PE pathogenesis. The expression of miR‐19a‐3p, PSG10P, and IL1RAP was examined in PE and normal placentas using RT‐qPCR. An in vitro experiment was performed in human trophoblast HET8/SVneo and TEV‐1 cells cultured in normoxic and hypoxic conditions. MiR‐19a‐3p targets were identified using Targetscan, miRanda, and PicTar analysis as well as luciferase reporter assays. The mouse model of PE was conducted using sFlt‐1 for in vivo tests. Lower levels of miR‐19a‐3p, but higher levels of PSG10P and IL1RAP were observed in PE placentas and the trophoblast cells in hypoxia. Luciferase reporter assays confirmed that PSG10P and IL1RAP were both direct targets of miR‐19a‐3p. Exposure to hypoxia inhibited cell viability, migration, and invasion of HET8/SVneo and TEV‐1 cells. Knocking out PSG10P and IL1RAP or overexpressing miR‐19a‐3p rescued the inhibition caused by hypoxia. In vivo experiments showed that IL1RAP promoted the expression of caspase‐3, a key apoptosis enzyme, but inhibited MMP9, which is responsible for degrading the extracellular matrix, suggesting a significant role of IL1RAP in cell proliferation, migration, and invasion. miR‐19a‐3p, PSG10P, and IL1RAP were all found to be involved in PE pathogenesis. With a common targeting region in their sequences, a regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway may contribute to PE pathogenesis during pregnancy.     

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth-associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant-pathogenic protozoan Plasmodiophora brassicae. Through a yeast two-hybrid assay, glutathione S-transferase pull-down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1-regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1-regulated genes in heterogeneous PBZF1-expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae-specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant-pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.     

<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> in its Environment

Plasmodiophora brassicae Wor. is viewed in this article from the standpoint of a highly evolved and successful organism, well fitted for the ecological niche that it occupies. Physical, chemical, and biological components of the soil environment are discussed in relation to their effects on the survival, growth, and reproduction of this microbe. It is evident that P. brassicae is well equipped by virtue of its robust resting spores for survival through many seasonal cycles. Germination is probably triggered as a result of signals initiated by root exudates. The resultant motile zoospore moves rapidly to the root hair surface and penetration and colonization follow. The short period between germination and penetration is one of greatest vulnerability for P. brassicae. In this phase survival is affected at the very least by soil texture and structure; its moisture; pH; calcium, boron, and nitrogen content; and the presence of active microbial antagonists. These factors influence the inoculum potential (sensu Garrett, 1956) and its viability and invasive capacity. There is evidence that these effects may also influence differentially the survival of some physiologic races of P. brassicae. Considering the interaction of P. brassicae with the soil environment from the perspective of its biological fitness is an unusual approach; most authors consider only the opportunities to destroy this organism. The approach adopted here is borne of several decades spent studying P. brassicae and the respect that has been engendered for it as a biological entity. This review stops at the point of penetration, although some of the implications of the environment for successful colonization are included because they form a continuum. Interactions with the molecular and biochemical cellular environment are considered in other sections in this special edition.

Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli

Using an antiserum of a patient with cutaneous manifestations of Lyme borreliosis we have isolated the gene encoding a 27 kDa protein antigen (P27) of Borrelia burgdorferi B29 from a λ-gt11 expression library. Nucleotide sequence analysis revealed that it is a basic protein of 248 amino acids with a typical prokaryotic leader sequence of 17 amino acid residues at the N-terminus of the proposed translation product. Biochemical investigations showed that P27 is a surface-exposed lipoprotein. From pulsed-field gel electrophoresis and subsequent Southern blot analysis it is evident that the p27 gene is located on a linear plasmid of a size of approximately 55 kb. It was over-expressed in Escherichia coli and the purified recombinant protein was used for biochemical and serological studies. Northern and Western blot analysis demonstrated that p27 is expressed in the European B. burgdorferi strain B29, but not in the American strain B31.     

Isolation of a Gene Greatly Expressed in Kluyveromyces Marxianus at High Temperature

Summary This research provides insight into the expression of thermotolerance-related genes in Kluyveromyces marxianus by differential display polymerase chain reaction (DD-PCR) techniques. Fourteen differential expressed sequence tags (ESTs) were observed and one of them, SHWBY10 was confirmed to be positive by reverse Northern blot analysis. The sequence has the GenBank Accession ID No. CD374838. DNA sequencing showed that it encoded a 279 bp ORF containing 92 amino acids. Analysis of protein sequence indicated that it has significant sequence homology with a peroxisomal protein product (gi 50309315) from Kluyveromyces lactis. This discovery suggests this gene may be related to yeast thermotolerance.     

Development of <Emphasis Type="Italic">Mamestra brassicae</Emphasis> and its solitary endoparasitoid <Emphasis Type="Italic">Microplitis mediator</Emphasis> on two populations of the invasive weed <Emphasis Type="Italic">Bunias orientalis</Emphasis>

The warty cabbage Bunias orientalis is an invasive pest in much of central Europe, including much of Germany since the 1980s, whereas in other countries, such as The Netherlands, it is a less common exotic species. Here, healthy larvae of Mamestra brassicae, which has been found feeding on B. orientalis plants in Germany, and larvae parasitized by one of its major larval endoparasitoids Microplitis mediator, were reared on both herbivore-induced and noninduced leaves of B. orientalis originating from single large populations growing in The Netherlands and central Germany. Herbivore performance was less negatively affected than parasitoid performance by differences in plant quality. Development times in both M. brassicae and Mi. mediator were shorter on Dutch than German plants and also shorter on noninduced than induced plants. Moreover, survival and body size of the parasitoid was more strongly affected by plant population and induction than survival of healthy M. brassicae. Chemical analyses of defensive secondary metabolites [glucosinolates (GS)] revealed that concentrations of the major GS sinalbin were constitutively expressed in German plants whereas they were induced in Dutch plants. However, in separate feeding bioassays in which preference for induced and noninduced leaves was compared separately, L3 instars of M. brassicae preferred noninduced German plants over Dutch plants but induced Dutch plants over German plants, revealing that changes in primary metabolites or an unidentified non-GS compound mediates population-related differences in plant quality. The results reveal asymmetric effects of plant quality in exotic plants on organisms in the second and third trophic level.