Microarray analysis of gene expression in Phytophthora capsici over-expressing an NMRA-like protein (PcNMRAL1, Phyca11_505845) during a time course infection assay of tomato (Solanum lycopersicum) leaves
Ontology highlight
ABSTRACT: Phytophthora capsici is a broad host range hemi-biotrophic pathogen which infects a wide range of agriculturally important crop plants. Infection with P. capsici is characterised by an early biotrophic phase which involves the formation of haustoria, followed by a necrotrophic phase later during infection. Previous data from our lab (Experiment E-MTAB-1295) revealed distinct transcriptional changes associated with biotrophy and necrotrophy, suggesting tight control and regulation of gene expression in P. capsici during the infection process, involving transcription factors and transcriptional regulators. A gene encoding for an NMRA-like protein (PcNMRAL1, Phyca11_505845) was identified as co-expressed with the biotrophy marker gene PcHmp1 encoding for a haustorial membrane protein. As this gene was found to be expressed during infection, and NMRA-like proteins are known to act as transcriptional regulators in fungi, this experiment aimed to investigate the effect of over-expression of PcNmrAL1 on gene expression in P. capsici (LT6535) during the early phase of infection (up to 24 hours post inoculation) on tomato leaves. P. capsici expressing the tdTomato fluorescence protein was used as a control strain. The results suggest PcNMRAL1 regulates the infection process by induction of biotrophy-related genes and suppression of necrotrophy-related genes. Tissue was sampled at 8, 16 and 24 hours post inoculation (hpi) for in planta stages, and at 24 hours for the in vitro mycelial stage.
Project description:Phytophthora species are a destructive group of filamentous plant pathogens, which have a global distribution and devastating effect on a wide range of plants important to agriculture and natural ecosystems. Throughout the infection cycle, Phytophthora secrete an effector repertoire into its host to inhibit or counter defence associated compounds, lytic enzymes and intracellular processes required for immunity. Despite the advent of genome sequences of both host and microbe however, little is known about the signal interplay between host and pathogen during infection. Here we explore and report on the association between the hemi-biotrophic broad host range pathogen Phytophthora capsici and tomato. Infection assays reveal a distinct hemi-biotrophic infection cycle, featuring haustoria formation early in infection, followed by necrotrophy in the lateinfection stages. We assessed gene expression changes during infection in both P. capsici and tomato and unveil distinct changes in both host and pathogen transcriptomes, associated with biotrophy and the subsequent switch to necrotrophy. These results suggest dynamic but highly regulated transcriptional programmes that underpin P. capsici hemi-biotrophy. Our results provide new detail on coordinate transcriptional reprogramming during infection and sheds light on the basic processes that accompany hemibiotrophy. Please note the timepoint is hours post inoculation (hpi) of P capsici zoospores on detached tomato leaves. Where it is na (not applicable), these are in vitro cultures of P capsici only.
Project description:The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at 10 different developmental and infection stages based on a 3'-tag digital gene expression (DGE) protocol. More than 90 million clean sequence tags were generated and compared to the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A comparison of the whole-library genes expression patterns suggested four groups: 1) mycelia and zoosporangia (MY and SP); 2) zoospores and cysts (ZO and CY); 3) germinating cysts (GC); 4) five infection site libraries (IF1.5 to IF24h). The libraries from the different groups showed major transitional shifts in gene expression. From the ten libraries, 722 gene expression pattern clusters were obtained and the top 16 ones, containing more than half of the genes, comprised enriched genes with different functions including protein localization, triphosphate metabolism, signaling process, and non-coding RNA metabolism. An evaluation of the average expression level of 30 pathogenesis related gene families revealed that most were infection induced, but with diverse expression patterns and levels. A web-based server named the Phytophthora Transcriptional Database (PTD) has been established. The five axenically grown stages were mycelia (MY), zoosporangia (SP), zoospores (ZO), cysts (CY), and germinating cysts (GC). The five infection stages, 1.5, 3, 6, 12 and 24 h after inoculation onto susceptible soybean leaf tissues (IF1.5h to IF24h).
Project description:Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease. The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana, and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy. The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom-designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages. 10 samples were used in this experiment.
Project description:Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these critical stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle, through an analysis of RNA from hyphae, sporangia, cleaving sporangia, motile zoospores, and germinated zoospore cysts. Keywords: Developmental study Gene expression in non-sporulating hyphae, asexual sporangia, sporangia undergoing cleavage, swimming zoospores, and germinated cysts containing appressoria were characterized using isolate 88069. Several strategies controlled for experimental error. Firstly, two replicate hybridizations were used for each tissue type. Secondly, RNAs for the two replicates were prepared by research groups in North America and/or Europe. Thirdly, RNA for each hybridization was pooled from two or three independent cultures made by that group. Finally, before using an RNA sample in an experiment the expression of selected well-characterized genes was measured to ensure that the tissue was properly prepared.
Project description:Samples were isolated from six different P. infestans life stages (hyphae, sporangia, zoospores, cysts, germinated cysts and appressoria).
Project description:Transcriptome of 3 developmental stages of Colletotrichum graminicola during infection of Zea mays leaf sheaths 3 biological replicates per stage. The three stages are: pre-penetration appressoria (PA), early biotrophic phase (BP), and the switch from biotrophy to necrotrophy (NP). Each biological replicate of the first stage, the pre-penetration appressoria, was sequenced to a 2-fold greater depth due to its lower representation in the samples.
Project description:To gain novel molecular insights into quantitative late blight resistance, we performed a high-resolution quantitative analysis of gene expression using potato cultivars with contrasting SNP alleles at the StAOS2 locus associated with maturity corrected resistance (MCR). SuperSAGE samples were generated from uninfected and infected plants of the selected genotypes under controlled environmental conditions. Genotypes were pooled to reduce the influence of the genetic background on the transcriptome. Nine SuperSAGE samples were prepared from artificially inoculated plants in a growth chamber using total RNA of the pooled 14, 6 and 9 genotypes in groups A1, A2 and B2, respectively, at three infection time points T0, T1 and T2. Combining the tag counts of both NlaIII and DpnII libraries resulted in 1.1 to 6.2 million tags per sample. Of total 266361 unique tags (unitags), 52.6% matched to the potato genome sequence when up to three mismatches per 26 base pairs were allowed, and 23.3% matched without mismatch. Fifteen pair wise comparisons were performed between the nine SuperSAGE samples to identify transcripts that were differentially expressed in response to infection (six comparisons) or between three genotype pools at the infection time points T0, T1 and T2 (nine comparisons). The number of unitags per comparison ranged from 127 000 to 182 000 (average 158 000). Between 2100 and 11800 tags were differentially expressed in pair wise comparisons, depending on arbitrary cut-off p-values for a significant difference. The highest number of differences was observed for the comparison between genotype pools A1 and A2 one day after infection (A1-T1 vs A2-T1), and the lowest between genotype pools A2 and B2 two days after infection (B2-T2 vs A2-T2). The number of differences in response to infection and between genotype pools even before infection (T0) was in the same order of magnitude. Based on the annotations in the DFCI potato gene index, in the potato genome and in few cases by BLASTX searches against the protein database at NCBI, transcripts that showed reproducible differential expression over the infection time course or between genotype pools A1, A2 and B2 were grouped in 16 functional categories, with overlaps between categories. Genes with genotype dependent, constitutive differential expression provide excellent targets for developing novel diagnostic markers for breeding cultivars with improved quantitative resistance to late blight and possibly other biotic and abiotic stresses. Relevant in this respect appear, besides numerous genes of unknown or ill-defined function, genes with known function involved in stress responses, photosynthesis, protein biosynthesis, protein degradation via the 26S proteasome, transport of proteins, lipids, ions and other small molecules, cell wall structure and many others. Eighteen SuperSAGE libraries were constructed based on nine leaf samples from one infection experiment. For each time point (T0, T1 and T2) one leaflet each of 14, 6 and 9 SL genotypes in genotypic groups A1, A2 and B2, respectively, were pooled. Frozen pooled leaf tissue was powdered in a CryoMill. SuperSAGE libraries were generated at GenXPro GmbH (Frankfurt, Germany) essentially as described (Matsumura et al. 2010). To prevent amplification biases the TrueQuant technology was applied as described by B. Rotter (Patent application Nr. WO2009152928). Besides NlaIII (recognition site: 5M-bM-^@M-^Y-CATG-3M-bM-^@M-^Y), DpnII (recognition site: 5M-bM-^@M-^Y-GATC-3M-bM-^@M-^Y) was used as second anchoring enzyme, to capture transcripts without a NlaIII site. Therefore, the 26 bp tags carry either CATG or GATC at their 5M-bM-^@M-^Y end. The libraries were pooled and sequenced by Solexa/Illumina technology (Illumina, Inc., USA).
Project description:This experiment contains Phytophthora sojae samples and RNA-seq data from experiment E-GEOD-29561 (https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-29651/) to understand gene expression during the P. sojae life cycle. The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at 5 different developmental stages: mycelia (MY), zoosporangia (SP), zoospores (ZO), cysts (CY) and germinating cysts (GC); based on a 3'-tag digital gene expression (DGE) protocol. More than 90 million clean sequence tags were generated and compared to the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A web-based server named the Phytophthora Transcriptional Database (PTD) has been established.
Project description:Colletotrichum graminicola is a hemibiotrophic fungal pathogen that causes maize anthracnose disease. It progresses through three recognizable phases of pathogenic development *in planta*: melanized appressoria that form on the host surface prior to penetration; biotrophy, characterized by colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development. An improved filtering algorithm and a Mixed Effects Generalized Linear Model (GLM) were developed and applied to an existing Illumina transcriptome dataset, substantially increasing the statistical power of the analysis of *C. graminicola* gene expression during infection and colonization. Additionally, the *in planta* transcriptome of the wild-type was compared with that of a mutant strain impaired in the establishment of biotrophy, allowing detailed dissection of events occurring specifically during penetration, and during early versus late biotrophy. Results indicated that there is a continuum of activities that occur during colonization of maize by *C. graminicola*, and that boundaries drawn between the three recognizable phases are artificial. More than 2000 fungal genes were differentially transcribed in waves during appressorial maturation, penetration, and colonization. Secreted proteins and membrane receptors were over-represented among the differentially expressed genes, suggesting that the fungus engages in an intimate and dynamic conversation with the host, beginning prior to penetration. This communication process is likely to involve reception of plant signals that trigger subsequent developmental progress in the fungus, as well as the production of signals that induce responses in the host. Later phases of biotrophy were more similar to necrotrophy, with increased production of secreted proteases, inducers of plant cell death, hydrolases, and membrane bound transporters for the uptake and egress of potential toxins, signals, and nutrients. The differentially expressed genes could be used as landmarks to more accurately identify developmental progress in compatible versus incompatible interactions involving genetic variants of both host and pathogen.
Project description:Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. However, all cultivated European grapevine varieties are susceptible to P. viticola and the resistance needs to be introduced from other Vitaceae. Segregating populations derived from Muscadinia rotundifolia, a species closely related to Vitis, lead to the identification and mapping of two resistance genes, named Rpv1 and Rpv2. The macroscopic phenotypes of the resistance mediated by the two loci are different. Rpv2 plants completely inhibit P. viticola sporulation and produce very small necrotic lesions. In contrast, Rpv1 plants allow a rather limited but visible sporulation of P. viticola. The aim of the study is to understand the gene expression changes associated with downy mildew resistance mediated by these two loci. Gene expression patterns after P. viticola inoculation or mock inoculation are compared in incompatible (resistant plants bearing Rpv1 or Rpv2 locus) and compatible (susceptible plants with no resistance loci) interactions, using the Vitis vinifera GeneChip from Affymetrix. In order to limit the effect of the genetic background, the plant material consists in three pools of genotypes called B, C and D, respectively corresponding to partially resistant plants (Rpv1+/Rpv2-), totally resistant plants (Rpv1-/Rpv2+), and susceptible plants (Rpv1-/Rpv2-) derived from a segregating population. Each pool consists in 3 different genotypes. Plant leaf discs were inoculated with P. viticola sporangium suspension (i) or mock-inoculated with water (ni) and analysed 6 hours post-inoculation. For each experimental condition, we performed two biological replicates. Experiment Overall Design: 3 plant genotypes were analyzed: Rpv1+/Rpv2- (B), Rpv1-/Rpv2+ (C), and Rpv1-/Rpv2- (D). Plants were either inoculated with P. viticola (i) or mock-inoculated with water (ni). Biological replicates were performed for each experimental condition.