Differential gene expression in Phytophthora infestans during pathogenesis on potato = [Differentiele genexpressie in Phytophthora infestans tijdens de pathogenese op aardappel]

The plant pathogenic fungus Phytophthora infestans (Mont.) de Bary is the causal agent of potato late blight, the most important and devastating fungal disease of potato. The molecular and cellular processes involved in pathogenicity of this hemibiotrophic fungus are poorly understood. The aim of the research described in this thesis was to gain more insight in these processes. A novel and unbiased approach was explored to isolate P . infestans genes which might have a function in pathogenesis. The approach was based on the assumption that the expression of pathogenicity genes of P . infestans , i.e. genes which are essential for the establishment and maintenance of basic compatibility or for the increase of disease severity, is specifically induced or significantly increased during pathogenesis. The strategy implies the isolation of in planta induced ( ipi ) genes by differential screening of a genomic library of P.infestans , subsequent characterization of their gene products, and elucidation of their function in pathogenesis.By differential screening, nine distinct in planta induced genes were isolated, i.e. ipiA , ipiB , ipiC , ipiD , ipiJ1 , ipiJ2 , ipiN , ipiO and ipiQ . Expression studies revealed that the ipiB and ipiO genes have a transient expression pattern during pathogenesis with the highest levels in early stages of the interaction. The seven other ip i-genes show a continuous 5-10 fold increase in expression level during growth of the fungus in the host.The P.infestans genes ipiA , ipiB , ipiC and ipiO were further characterized. Two of these appeared to encode ubiquitous proteins: ipiA , which was renamed ubi3R , encodes ubiquitin and ipiC , renamed calA , codes for calmodulin. Both proteins are highly conserved in eukaryotic organisms and have been shown to play important roles in basic cellular processes such as selective degradation of abnormal proteins (ubiquitin) and signal transduction (calmodulin). This is the first time that an increased expression of ubiquitin and calmodulin genes is found in association with pathogenicity of a plant pathogenic fungus. However, the significance of increased ubiquitin and calmodulin levels in P.infestans during pathogenesis remains to be resolved. The ipiB gene belongs to a gene family consisting of at least three highly homologous genes, ipiB1 , ipiB2 and ipiB3 , which are clustered on the genome in a head-to-tail arrangement. The ipiB genes encode novel glycine-rich proteins (GRPs) of 301 (IPI-B1), 343 (IPI-B2), and 347 (IPI-B3) amino acids (aa) respectively. The primary structure of the IPI-B proteins is composed of a N-terminal signal sequence followed by a large, highly repetitive glycine-rich domain. The glycine-rich domain is predominantly composed of two repeats with the core sequences A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G, respectively. The characteristic primary structure is also found in plant GRPs of which some have been shown to be associated with the plant cell wall. The P. infestans genome contains two ipiO genes, ipiO1 and ipiO2 , which are closely linked and arranged in an inverted orientation. The ipiO genes encode two nearly identical 152 aa proteins which have no homology with any known sequence. As the IPI-B proteins, the IPI-O proteins contain a N-terminal signal sequence suggesting that they are excreted. In addition, the IPI-O proteins have a putative N-glycosylation site and a typical Arg-Gly-Asp (RGD) tripeptide motif. The RGD tripeptide is conserved in several mammalian extracellular adhesion proteins in which it functions as a cell attachment motif. Whether the IPI-O proteins have adhesion properties is unknown.The four in planta induced P . infestans genes ubi3R , calA , ipiB and ipiO , are among the first of the limited group of oomycetous genes to be isolated and sequenced. As in most known oomycetous genes, the coding sequence of these genes is not interrupted by introns. Within 100 nucleotides upstream of their ATG start codon, a conserved motif matching the consensus sequence GCTCATTYYNCA(A/T)TTT was identified. Comparison of the 5' nontranscribed regions of eight distinct oomycetous genes revealed that this sequence motif surrounds the transcription start point of the majority of these genes, suggesting that oomycetous genes have a GCTCATTYYNCA(A/T)TTT sequence preference for transcription initiation.The expression of the ipiB and ipiO genes was studied in more detail. The ipiB genes as well as the ipiO genes are expressed in germinating cysts prior to host penetration. During pathogenesis both genes are transiently expressed. The highest mRNA levels are found in early stages of infection. Also during initial stages of interactions of P.infestans with a racespecific resistant host and the nonhost Solanum nigrum L., the expression of the ipiB and ipiO genes is induced. During growth of the fungus in vitro, nutrient deprivation appeared to be a strong stimulus for the induction of ipiB and ipiO gene expression. For several other pathogenesis related genes characterized in fungal plant pathogens it has been shown that nutrient starvation induces their expression. Whether starvation conditions mimic the nutrient condition encountered upon infection and, as such, function as a general stimulus for the activation of pathogenicity genes remains to be established.Manipulation of gene expression in P.infestans is an important tool to study gene function. The possibility to manipulate gene expression in P. infestans by anti-sense RNA was tested. First, the promoters of five oomycetous genes, among which the P.infestans ubi3R gene, were fused to the reporter β-glucuronidase ( GUS ) gene of E. coli and the activity of the promoters was determined in transient expression assays as well as in stable transformants. The hsp70 and ham34 genes of Bremia lactucae appeared to have the highest activity. When the GUS gene in the anti-sense orientation fused to the ham34 promoter, is introduced in transgenic P.infestans containing sense GUS constructs, the accumulation of GUS is effectively inhibited. This indicates that the anti-sense technique is suitable to study gene function in P.infestans.</em

Использование макромолекулярных бактериоцинов для селекции популяционных диссоциантов Erwinia carotovora subsp. сarotovora

Запропоновано метод специфiчної селекцiї за допомогою каротоворицинiв типу фагових хвостових вiдросткiв, який дозволяє проводити ефективний вiдбiр популяцiйних дисоцiантiв рiзних типiв Erwinia carotovora. Показано, що спонтаннi дисоцiанти i дисоцiанти, одержанi за допомогою селекцiї бактерiоцинiв, вiдрiзняються колонiально-морфологiчними, ростовими характеристиками i такою бiохiмiчною ознакою, як синтез позаклiтинної пектатлiази. Встановлено, що змiна поверхневих структур дисоцiантiв приводить до змiни апаратiв секрецiї II i III типу — основних факторiв патогенностi пектолiтичних ервiнiй. Одержанi результати є передумовою для вивчення напряму i механiзму дисоцiацiї у практично важливої бактерiї Е. carotovora.We propose a method of specific selection of dissociants of varions strains of Erwinia carotovora, by using carotovoricins that are identical to the phage tails. It is shown the spontaneous dissociants and those which were obtained by the selection of bacteriocins differ by morphological and growing features and by the synthesis of outer pectatlyase. It is found that the modifications of the surface structures of dissociants are the reason for the change of mode secretions of II and III types — the main factors of pathogenicity of pectolytic erwinia. The obtained data are the prerequisite for studying the direction and the mechanism of dissociation of such practically important bacteria as E. carotovora

Ігор Степанович Флюнт - доктор медичних наук

23 грудня 2003 року на засіданні спеціалізованої вченої Д 26.198.01 при Інституті фізіології ім. О.О. Богомольця НАН України успішно захистив докторську дисертацію за спеціальністю 14.03.04 - патологічна фізіологія провідний бальнеолог України, лауреат премії ім. Т. Торосєвича в галузі бальнеології, член Нью-Йоркської АН, член Ради Асоціації учених міста Трускавця, керівник трускавецької групи клінічної бальнеології та фітотерапії Інституту фізіології ім. О.О. Богомольця, начмед санаторію “Весна” ЗАТ ЛОЗ “Трускавецькурорт” Ігор Степанович ФЛЮНТ

Развитие ионизационного монитора поперечного сечения протонного пучка линейного ускорителя ИЯИ РАН

Для обеспечения прозрачных измерений поперечного сечения и профилей токовых импульсов в широком диапазоне энергий и амплитуд разработан и установлен на ускорителе специальный ионизационный монитор поперечного сечения (ИМПС) на остаточном газе. ИМПС оборудован зеркально-линзовым трактом для транспортировки изображения пучка от детектора до ТВ-камеры и защиты ПЗС-матрицы и электроники ТВ-камеры от бомбардировки нейтронами и γ-квантами. В работе приводится схема и описание датчика, а также некоторые детали программного и аппаратного обеспечения системы съема и обработки изображений. Представлены полученные результаты измерений импульсного тока протонов.Для забезпечення прозорих вимірів поперечного переріза й профілів струмових імпульсів у широкому діапазоні енергій і амплітуд розроблений і встановлений на прискорювачі спеціальний іонізаційний монітор поперечного переріза (ІМПС) на залишковому газі. ІМПС обладнаний дзеркально-лінзовим трактом для транспортування зображення пучка від детектора до ТВ-камери і захисту Пзс-матриці й електроніки ТВ-камеры від бомбардування нейтронами і γ-квантами. У роботі приводиться схема й опис датчика, а також деякі деталі програмного й апаратного забезпечення системи знімання й обробки зображень. Представлено отримані результати вимірів імпульсного струму протонів.To provide non-intercepting measurements of beam pulse transverse section and profile the special residual gas ion transverse section monitor (ITSM) for wide energy and amplitude range is developed and installed on the accelerator. ITSM is provided by lens-mirror line for transport beam image from the detector to TV camera and saving CCD and electronics of TV camera from neutron and γ hitting. The ITSM functioning details and image processing system are described. The available results of beam pulse measurements are presented

Induced plant responses to microbes and insects

Plants are members of complex communities and interact both with antagonists and beneficial organisms. An important question in plant defense-signaling research is how plants integrate signals induced by pathogens, insect herbivores and beneficial microbes into the most appropriate adaptive response. Molecular and genomic tools are now being used to uncover the complexity of the induced defense signaling networks that have evolved during the arms races between plants and the other organisms with which they intimately interact. To understand the functioning of the complex defense signaling network in nature, molecular biologists and ecologists have joined forces to place molecular mechanisms of induced plant defenses in an ecological perspective. In this Research Topic, we aim to provide an on-line, open-access snapshot of the current state of the art of the field of induced plant responses to microbes and insects, with a special focus on the translation of molecular mechanisms to ecology and vice versa. We will collect Original Research and Review papers on the topic, but also other article types, such as Methods and Opinions are welcome

Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance

In Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers jasmonate (JA)- and ethylene (ET)-dependent induced systemic resistance (ISR) that is effective against different pathogens. Arabidopsis genotypes unable to express rhizobacteria-mediated ISR against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) exhibit enhanced disease susceptibility towards this pathogen. To identify novel components controlling induced resistance, we tested 11 Arabidopsis mutants with enhanced disease susceptibility (eds) to pathogenic P. syringae bacteria for WCS417rmediated ISR and pathogen-induced systemic acquired resistance (SAR). Mutants eds4-1, eds8-1 and eds10-1 failed to develop WCS417r-mediated ISR, while mutants eds5-1 and eds12-1 failed to express pathogen-induced SAR. Whereas eds5-1 is known to be blocked in salicylic acid (SA) biosynthesis, analysis of eds12-1 revealed that its impaired SAR response is caused by reduced sensitivity to this molecule. Analysis of the ISR-impaired eds mutants revealed that they are non-responsive to induction of resistance by methyl jasmonate (MeJA) (eds4-1, eds8-1 and eds10-1), or the ET precursor 1- aminocyclopropane-1-carboxylate (ACC) (eds4-1 and eds10-1). Moreover, eds4-1 and eds8-1 showed reduced expression of the plant defensin gene PDF1.2 after MeJA and ACC treatment, which was associated with reduced sensitivity to either ET (eds4-1) or MeJA (eds8-1). Although blocked in WCS417r-, MeJA- and ACC-induced ISR, eds10-1 behaved normally for several other responses to MeJA or ACC. The results indicate that EDS12 is required for SAR and acts downstream of SA, whereas EDS4, EDS8 and EDS10 are required for ISR acting either in JA signalling (EDS8), ET signalling (EDS4), or downstream JA and ET signalling (EDS10) in the ISR pathway

Cross-talk between signaling pathways leading to defense against pathogens and insects

In nature, plants interact with a wide range of organisms, some of which are harmful (e.g. pathogens, herbivorous insects), while others are beneficial (e.g. growth-promoting rhizobacteria, mycorrhizal fungi, and predatory enemies of herbivores). During the evolutionary arms race between plants and their attackers, primary and secondary immune responses evolved to recognize common or highly specialized features of microbial pathogens (Chisholm et al., 2006), resulting in sophisticated mechanisms of defense

Micro-organismen beschermen planten tegen rupsenvraat

Samenvatting van de voordracht te houden op 30 november 2005 tijdens de Najaarsvergadering van de KNPV (Koninklijke Nederlandse Plantenziektekundige Vereniging). Onderzoek naar inductie van resistentie in Arabidopsis tegen vraat van rupse

Two-way plant mediated interactions between root-associated microbes and insects: from ecology to mechanisms

Plants are members of complex communities and function as a link between above- and below-ground organisms. Associations between plants and soil-borne microbes commonly occur and have often been found beneficial for plant fitness. Root-associated microbes may trigger physiological changes in the host plant that influence interactions between plants and aboveground insects at several trophic levels. Aboveground, plants are under continuous attack by insect herbivores and mount multiple responses that also have systemic effects on belowground microbes. Until recently, both ecological and mechanistic studies have mostly focused on exploring these below- and above-ground interactions using simplified systems involving both single microbe and herbivore species, which is far from the naturally occurring interactions. Increasing the complexity of the systems studied is required to increase our understanding of microbe-plant-insect interactions and to gain more benefit from the use of non-pathogenic microbes in agriculture. In this review, we explore how colonization by either single non-pathogenic microbe species or a community of such microbes belowground affects plant growth and defense and how this affects the interactions of plants with aboveground insects at different trophic levels. Moreover, we review how plant responses to foliar herbivory by insects belonging to different feeding guilds affect interactions of plants with non-pathogenic soil-borne microbes. The role of phytohormones in coordinating plant growth, plant defenses against foliar herbivores while simultaneously establishing associations with non-pathogenic soil microbes is discussed

Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes

Root-associated microbiota can protect plants against severe disease outbreaks. In the model-plant Arabidopsis thaliana, leaf infection with the obligate downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) results in a shift in the root exudation profile, therewith promoting the growth of a selective root microbiome that induces a systemic resistance against Hpa in the above-ground plant parts. Here we show that, additionally, a conserved subcommunity of the recruited soil microbiota becomes part of a pathogen-associated microbiome in the phyllosphere that is vertically transmitted with the spores of the pathogen to consecutively infected host plants. This subcommunity of Hpa-associated microbiota (HAM) limits pathogen infection and is therefore coined a “resistobiome”. The HAM resistobiome consists of a small number of bacterial species and was first found in our routinely maintained laboratory cultures of independent Hpa strains. When co-inoculated with Hpa spores, the HAM rapidly dominates the phyllosphere of infected plants, negatively impacting Hpa spore formation. Remarkably, isogenic bacterial isolates of the abundantly-present HAM species were also found in strictly separated Hpa cultures across Europe, and even in early published genomes of this obligate biotroph. Our results highlight that pathogen-infected plants can recruit protective microbiota via their roots to the shoots where they become part of a pathogen-associated resistobiome that helps the plant to fight pathogen infection. Understanding the mechanisms by which pathogen-associated resistobiomes are formed will enable the development of microbiome-assisted crop varieties that rely less on chemical crop protection