Unknown,Transcriptomics,Genomics,Proteomics

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Differential expression of miRNAs in Brassica napus root following infection with Plasmodiophora brassicae

ABSTRACT: Canola (oilseed rape, Brassica napus L.), is susceptible to infection by the biotrophic protist Plasmodiophora brassicae, the causal agent of clubroot. To understand the roles of microRNAs (miRNAs) during the post-transcriptional regulation of disease initiation and progression, we have characterized the changes in miRNA expression profiles in canola roots during clubroot disease development and have compared these to uninfected roots. Two different stages of clubroot development were targeted in this miRNA profiling study: an early time of 10-dpi for disease initiation and a later 20-dpi, by which time the pathogen had colonized the roots (as evident by visible gall formation and histological observations). P. brassicae responsive miRNAs were identified and validated by qRT-PCR of miRNAs and the subsequent validation of the target mRNAs through starBase degradome analysis, and through 5M-bM-^@M-^Y RLM-RACE. This study identifies putative miRNA-regulated genes with roles during clubroot disease initiation and development. Putative target genes identified in this study included: transcription factors (TFs), hormone-related genes, as well as genes associated with plant stress response regulation such as cytokinin, auxin/ethylene response elements. The results of our study may assist in elucidating the role of miRNAs in post-transcriptional regulation of target genes during disease development and may contribute to the development of strategies to engineer durable resistance to this important phytopathogen. In this miRNA-microarray experiment a total of 4 samples were analyzed with their 3 biological replicates. In which 2 samples C 10 DAY and C 20 DAY was used as referrence contols.

ORGANISM(S): Brassica napus

SUBMITTER: Shiv Verma

PROVIDER: E-GEOD-51590 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Clubroot of Brassicaceae, an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant loss to seed yield. The pathogen is continuously evolving and new pathotypes are emerging, this necessitates the development of novel resistant canola cultivars to manage the disease effectively. Given that proteins play a crucial role in majority of biological processes and molecular functions, the identification of differentially abundant proteins (DAP) using proteomics information is an attractive approach to understand the plant-pathogen interactions as well as in the future development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7-, 14-, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to LC-MS/MS analysis. A total of 937 proteins demonstrated a significant (q < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly (q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of the differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways in response to the pathogen. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 74 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in four chromosomes including chromosomes A3 and A8 were identified. In conclusion, these results have contributed to an improved understanding of the mechanisms that are involved in mediating response to P. brassicae in canola at the protein level.

Project description:The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested to investigate specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using Laser Microdissection and Pressure Catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected to contol plants. We were especially interested to elucidate the stage-specific hormonal network. The upregulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception was in many cases upregulated in enlarged cells and the central cylinder. This was confirmed by qPCR and mutant analysis of the BR receptor mutant bri1-6, which exhibited less severe gall formation than the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using this method of single cell preparation combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner. Transcription profiling was performed in isolated Arabidopsis thaliana root cells harboring different developmental stages of Plasmodiophora brassicae at two time points after inoculation (dai) (14 and 21 dai), as well as in infected central cylinder tissue from roots at 14 dai (days after inoculation). Control samples were taken from uninfected roots. Host cells were dissected from paraffin embedded roots using Laser Microdissection and Pressure Catapulting (LMPC). 8 samples have been analyzed.