Transcription profiling by array of vdh1 mutant and wild type samples of soilborne fungus Verticillium dahliae
ABSTRACT: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequentially, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae.
Project description:Verticillium dahliae is a soilborne fungus that causes wilt disease in plants. The microsclerotia of V. dahliae produce infectious hyphae that give rise to primary infections. In this study, RNA-seq libraries were prepared from microsclerotia (MS)-producing cultures of V. dahliae (ave = 52.23 million reads), and those not producing microsclerotia (NoMS, ave = 50.58 million reads) and analyzed for differential gene expression.
Project description:Eggplant is susceptible to fungal wilts caused by Fusarium oxysporum f. sp. melongenae and Verticillium dahliae. Wild relatives represent a good source of resistance and ILs have been obtained through introgression of the Rfo-sa1 locus conferring resistance to Fusarium oxysporum from the allied species S. aethiopicum into cultivated eggplant. In this work, a deep phenotypical characterization was performed according to the progression of symptoms along the stem and the disease severity in leaves. This analysis showed that the Fom -resistant ILs carrying introgression of the Rfo-sa1 locus displayed significantly improved tolerance to Verticillium attack after a preliminary inoculation with F. oxysporum. This positive effect was particularly evident when Verticillium inoculation was done simultaneously or after the Fusarium one. Transcript profiling carried out using a combination of SSH, microarray and qRT-PCR analyses from inoculated roots with a selected combinations of fungal pathogens enabled the identification of 164? differentially expressed genes at least in one condition. Overall, our results highlighted a number of candidate genes putatively involved in early defence responses or signalling pathways activated upon infection of eggplant either with Fom and Vd and thus leading to a broad Rfo-sa1 mediated tolerance against both these wilt pathogens. Overall design: S. melongena roots mock inoculated, Fusarium oxysporum f. sp. Melongenae inoculated, Verticillium dahlie inoculated and inculated by both Fusarium and Verticillium have been collected in biological triplicate at 0, 4 and 8 hours post infection to evaluate expression levels with an array contructed from ESTs and RAD tags
Project description:Arabidopsis plants were challenged with Ralstonia solanacearum isolate BCCF401 and expression profiles investigated during early and late wilt symptom development. Keywords: Disease state analysis A direct comparison was performed. Two replicate biological experiments were performed and two technical replicates with dye swap were included in each biological replicate.
Project description:Transcriptome sequencing is a powerful approach to globally delineate both the transcriptional and post-transcriptional genome regulation in human and other higher eukaryotes. This study sequenced the transcriptomes of two pathogenicity-differential strains of the plant wilt pathogen Verticillium dahliae. Although they showed no growth difference in vitro, hundreds of V. dahliae genes including those synthesizing aflatoxin were preferentially expressed at in the high-virulence strain. Using both the Pfam and GO annotation strategies, some of these putative virulence genes were ambiguously clustered into several known pathogenic mechanisms including hydrophobins secreted from fungal cells and acting as phytotoxin, biosynthesis of melanin protecting the fungal pathogen against host immune responses, membrane proteins of CFEM and major facilitator superfamily MFS1 with known functions in pathogenesis and multi-drug resistance, respectively. These results suggest that some pathogenicity pathways are pre-activated at the transcriptional level prior to the fungal infection of host plants. We developed two algorithms to confidently identify 1518 alternative splicing events in 1,259 Verticillium genes, representing 15.1% of multi-exonic genes. Among the events, 43.5% involving in novel splice sites were classified into nine AS types, the others belong to intron retention. Verticillium AS genes were exclusively enriched in the regulatory biological processes such as mycelium development, reproduction, morphogenesis, cell communication and signal transduction, predicting a primary function of AS regulation when the pathogen infecting its host. This work presents a transcriptome-wide approach for identifying the fungal virulence genes and pathogenicity mechanisms; both the methodology and mechanisms could be generally applicable to other fungal pathogens. Overall design: Transcriptome analysis of two Verticillium dahliae strains
Project description:Verticillium dahliae is a soil-borne vascular pathogen that causes severe wilt symptoms in a wide range of plants. Co-culture of the fungus with Arabidopsis roots for 24 hours induces many changes in the gene expression profiles of both partners, even before defense-related phytohormone levels are induced in the plant. Both partners reprogram sugar and amino acid metabolism, activate genes for signal perception and transduction, and induce defense and stress responsive genes. Furthermore, analysis of Arabidopsis expression profiles suggests a redirection from growth to defense. The plant and fungal genes that rapidly respond to the presence of the partner might be crucial for early recognition steps and the future development of the interaction. Thus they are potential targets for the control of V. dahliae-induced wilt diseases. Overall design: The aim of this study was to identify important targets regulated in both partners in the initial phase of interaction (24h). After 24 h of co-cultivation the fungus colonizes the plant root and first penetration of plant tissue by the fungus is visible while the vascular tissue of the plant is not yet colonized (pre-vascular phase).
Project description:Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry-based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) was used to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. We designed a peptide filter method enabling the identification of tryptic peptides derived from amyloidogenic and amyloid-associated proteins without additional tandem mass spectrometry. Utilizing the filter we found a universal peptide signature for amyloidoses independent from amyloid type and histoanatomical localization. Examining a validation cohort of cardiac biopsies including 66 amyloid and 31 non-amyloid cases, amyloidosis was diagnosed with high sensitivity and specificity. Furthermore, differences in the peptide composition of AL-lambda and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.
Project description:Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato. Two-color microarray comparison of two clones, three biological replicates of each clone, one dye swap technical replicate
Project description:C-type natriuretic peptide (CNP) has been recently identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanism mediating these effects are not completely understood. Here we demonstrate that CNP activates the p38 MAP kinase pathway in chondrocytes and that pharmacological inhibition of p38 blocks the anabolic effects of CNP in a tibia organ culture system. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We performed Affymetrix microarray analyses to identify CNP target genes in the organ culture system. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, potentially because cGMP-dependent kinases I and II, important transducers of CNP signaling and are expressed at much higher levels in these cells than in other areas of the tibia. One of the genes most strongly induced by CNP was the Ptgs2 gene, encoding Cox2. Real-time PCR confirmed that Cox2 expression was induced by CNP in hypertrophic chondrocytes, but surprisingly in a p38-independent manner. Moreover, Cox2 inhibition – in contrast to p38 inhibition - did not block the anabolic effects of CNP. In summary, our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral ossification, with potential implications for numerous skeletal diseases. Experiment Overall Design: Tibiae from E15.5 day old embryonic mice were isolated and cultured in minimal media in the presence of vehicle, BSA/HCl (1mM), or C-type natriuretic peptide, CNP (10-6M). On the sixth day of treatment cultured tibias were micro-dissected into the resting/proliferating, hypertrophic, and mineralized areas. Distinct zones from approximately 24 bones were pooled together, from which RNA was isolated using the Qiagen RNeasy Lipid Extraction Kit. Once the quality of total RNA from three independent trials was determined using the Agilent 2100 bioanalyzer, microarray analyses were performed at the London Regional Genomics Centre using MOE430_2.0 Affymetrix arrays. Results were analyzed using GeneSpring 7.2 software.