Compatible and incompatible infection of potato by nematode Globodera rostochiensis
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ABSTRACT: The cultivar Desirée and the breeding line SW93-1015 were challenged with potato cyst nematodes and transcriptomes analysed after 8h and 48 h.
Project description:In this experiment we evaluate the transcriptional responses of G pallida D383 1-2A to diverse hatching stimulations by pure Solanoeclepin A or by potato root exudate. We used cysts hydrated for 7 days in tap water for the experiment. Water for hydration was changed every day. After hydration approximately 13mg of cysts (50-100 cysts) were placed over Netwell inserts (Corning) with 74um mesh size. For this experiment we used 5 “technical” replicates, all performed in the same experiment. Worms underwent the treatment at the Laboratory of Nematology (WUR)(Spit lab) and RNA was extracted in the same lab using the Qiagen RNeasy Micro kit. RNA samples were measured by Nanodrop and Qubit and were sent to the Wageningen BioInformatics business unit. (Wageningen, The Netherlands, Dr. Sara Diaz Trivino’s team) on dry ice. Sequencing was performed by the DLO in Wageningen.
Project description:Rice false smut (RFS) is a kind of fungal disease transforming panicles and spikelets into greenish spore balls, caused by Ustilaginoidea virens. During artificial cultivation process, macroscopic exudates could be observed, which is a common feature in many kinds of fungi. We characterized the proteome of exudate associated with this plant pathogen. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to identify proteins in the pathogen exudate. A total of 685 proteins comprising 3,949 peptides were identified from the exudate. This study regarded the biological characteristics of U. virens as an entry point. By studying the protein components of the exudates of U. virens, it is helpful to better understand the occurrence and pathogenic mechanism of pathogen and provide a theoretical basis for the control of RFS.
Project description:In this project we sequenced smallRNAs to see how both BCN (H.schachtii) and PCN (G. rostochiensis line 19 and 22) react to different viruses. After adapter removal, small RNA analysis was performed on sequences from 18 to 28 nt in length. Also the smallRNAs are mapped to the reference genomes of the nematodes to look for smallRNAs.
Project description:TRD hatched preparasitc juveniles of line-22 from Globodera rostochiensis were collected and total RNA was isolated as described under ”Materials and methods” in the manuscript. Library prep was conducted using a ribosome depletion method. The same protocols and methods applies to Ro1-line19: SRR16693885
Project description:The differentiation of specialized feeding sites in Arabidopsis root cells in response to nematode infestation involves substantial cellular reprogramming of host cells that is not well characterized at the molecular level. Expression data was generated from Arabidopsis root cells undergoing giant cell formation due to nematode infestation and from non-infested control root cells. Cells were laser captured 14 and 21 days after infestation. Samples, collected 14 days post infestation, consisted of three biological replicates per treatment (control root cells or giant cells). RNA samples were isolated from ~150 control cells or from ~80 giant cells using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, USA). RNA amplifications were carried out with the NuGEN WT-Ovation Pico kit. GSM546568-GSM546577: Control and giant cells collected 21 days post infestation.
Project description:Background; Heterodera schachtii is an economically important plant parasitic nematode that forms a syncytium from a cell superficial to the formed vascular bundle by progressive recruitment of other cells into the structure. The pattern of plant gene expression changes dramatically inside the syncytium. The pathogen probably plays a major role in defining the plant response by choice of initial plant cell during precise behaviour in planta and/or by the secretions it releases. The modified plant cells enable a high feeding rate by the female nematode so enhancing its rate of development and subsequent daily egg production. Arabidopsis is widely used as a model plant to characterise molecular responses to nematodes (e.g. Sijmons et al., 1991 Plant J. 1:245-254.). A complete overview of the changes in plant gene expression when sedentary nematodes establish has not yet been gained using Arabidopsis or any other host plant. Experimental Approaches; Our initial studies will focus on the H. schachtii/Arabidopsis interaction. To assure reliable microarray screening care has been taken to minimise extraneous differences between samples (see "Growth conditions" section). At 21 days (Growth stage 3.2-3.5 Boyes et al., 2001 Plant Cell 13:1499-1510) Arabidopsis plants were challenged with rigorously sterilised, infective nematodes of H. schachtii as before (Urwin et al., (1997) Plant Journal 12: 455-461.). 35 sterile J2s were pipetted onto small ~0.5mm2 squares of sterile GF/A filter paper. The GF/A paper was left in direct contact with the zone of elongation on 3 lateral roots per plant for 48 hours. Control plants were mock inoculated with sterile water. Sections of root containing syncytia have been excised from the thin and transparent roots of Arabidopsis and collected into RNAlater solution (Ambion) at 21 days post infection (Growth Stage 6.1 Boyes et al. 2001). The female nematode has been removed with watch-maker's forceps. Equivalent sections of root have been harvested from non-infected plants. Material has been collected from c. 1000 plants for each of the two samples and the uninfected material serves as an internal control. Total RNA has been prepared from the reference and test root material using an RNeasy plant RNA preparation kit (Qiagen) according to methods required by GARNET.Some questions on the form are omitted as we are not using mutant or transgenic lines. This is our first application. Experimenter name = Peter Edward Urwin; Experimenter phone = 0113 343 3035/2909; Experimenter fax = 0113 343 3144; Experimenter address = Centre for Plant Science; Experimenter address = University of Leeds; Experimenter address = Leeds; Experimenter zip/postal_code = LS2 9JT; Experimenter country = UK Experiment Overall Design: 2 samples were used in this experiment
Project description:Plant-parasitic nematodes (PPN) need to be adapted to survive in the absence of a suitable host or in hostile environmental conditions. Various forms of developmental arrest (including desiccation, cryopreservation, hatching inhibition and dauer stages) are used by PPN in order to survive these conditions and spread to other areas. Potato cyst nematodes (PCN) (Globodera pallida and G. rostochiensis) are frequently in a dessicated state unhatched nematodes within the egg dispersal unit inside the cyst. Long term survival seems to be associated primarily with species that have a very restricted host range which requires surviving unhatched in the absence of the host for extended periods of time. This paper shows fundamental changes in the response of quiescent and diapaused eggs of G.pallida to hydration and following exposure to tomato root diffusate using microarray gene expression analysis from a broad set of genes. Surprisingly, many unique genes were activated in the population of diapaused eggs. Transport activity was activated in both quiescent and diapaused eggs; however, the transport function genes were very different between them. Hydrated quiescent and diapaused eggs were markedly different indicating differences in adaptation for long term survival.
Project description:Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser capture microdissection. Arabidopsis root cells undergoing giant cell formation due to nematode infestation and un-infested control root cells were laser captured and used to evaluate 2 amplification systems. One, NuGEN's WT-Ovation Pico amplification system, uses total RNA as starting material while the other, NuGEN's WT-One-Direct Amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The NuGEN WT-Ovation One-Direct system was less reproducible and more variable than the NuGEN WT-Ovation Pico system. The NuGEN WT-Ovation Pico Amplification kit resulted in the detection of thousands of genes differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the NuGEN WT-Ovation One-Direct Amplification kit. Evaluation of each amplification system consisted of three biological replicates per treatment (control root cells or giant cells) with one biological replicate split into three technical replicates for a total of 10 samples per amplification procedure. RNA samples for amplification with the NuGEN WT-Ovation Pico kit were isolated from 150 control cells or from 80 giant cells using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, USA). For amplification with the NuGEN WT-Ovation One-Direct kit, samples containing 150 control or 80 giant cells were collected directly into 2ul of the NuGEN lysis buffer. RNA amplifications were carried out according to the respective kitsâ?? protocols.
Project description:In this experiment we measured the transcriptional response of tomato plants (cv “Money maker”) when attacked belowground by the nematode M. inognita and, subsequently, aboveground by different (and common for this crop) biotic agents. Three weeks-old plants were exposed to nematodes for 5 days. At the fifth day the terminal leaflet of one of the first two true leaves was infected with the Cauliflower Mosaic Virus, or with the pathogen, or with the potato aphid Macrosiphum euphorbiae, or with the CMV-infected M. euphorbiae. For each belowground/aboveground combination treatment a set of control plants that received only the aboveground treatment was prepared. The infected leaflets of 5 biological replicates, each consisting of 1 plant, were collected 1, 2, 3, 4, 5 and 6 days after the onset of the aboveground treatment and flash frozen in liquid nitrogen. A different set of plants was used for every time point. Corresponding leaves from plants that did not receive any aboveground treatment (control) were selected and sampled as described above. Three biological replicates were selected among the five for RNA isolation. Total RNA was sent for sequencing to BGI Hong Kong.
Project description:Parallel genome-wide expression profile of soybean and soybean cyst nematode at three points during infection 2, 5, 10 days post-inoculation.