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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 5’ 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.

ORGANISM(S): Manihot esculenta Panicum virgatum Nasturtium officinale Hordeum vulgare Cenchrus ciliaris Physcomitrium patens Medicago truncatula Lupinus luteus Petunia x hybrida Oryza sativa Hordeum vulgare subsp. vulgare Oryza brachyantha Prunus salicina Phleum pratense Populus trichocarpa x Populus deltoides Rorippa indica Populus tremula Malcolmia maritima Picea sitchensis Populus trichocarpa Oenocarpus bataua Ipomoea nil Ricinus communis Phaseolus coccineus Nuphar advena Phaseolus vulgaris Prunus armeniaca Liriodendron tulipifera Oryza nivara Oryza rufipogon Pinus taeda Nicotiana sylvestris Nicotiana tabacum Prunus persica Picea glauca Brassica napus Lolium arundinaceum Cenchrus americanus Lactuca sativa Nicotiana benthamiana Lotus japonicus Oryza barthii Malus domestica Ipomoea batatas Populus tremuloides Peridictyon sanctum Populus tremula x Populus tremuloides Persea americana Sorghum bicolor

PROVIDER: GSE51590 | GEO | 2013/11/28

SECONDARY ACCESSION(S): PRJNA223665

REPOSITORIES: GEO

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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:Gall formation on the belowground parts of plants infected with Plasmodiophora brassicae is the result of extensive host cellular reprogramming. The development of these structures is a consequence of increased cell proliferation followed by massive enlargement of cells colonised with the pathogen. Drastic changes in cellular growth patterns create local deformities in the roots and hypocotyl giving rise to mechanical tensions within the tissue of these organs. Host cell wall extensibility and recomposition accompanies growth of the gall, influences pathogen spread and also pathogen life cycle progression. Demethylation of pectin within the extracellular matrix may play an important role in P. brassicae-driven hypertrophy of host underground organs. Through proteomic analysis of the cell wall we identified proteins accumulating in the galls developing on the underground parts of Arabidopsis thaliana plants infected with P. brassicae. One of the key proteins identified was the pectin methylesterase PME18; we further characterised its expression and conducted functional and anatomic studies in the knock-out mutant and used Raman spectroscopy to study the status of pectin in P. brassicae infected galls. We found that late stages of gall formation are accompanied with increased levels of Pectin Methylesterase 18 (PME18). We have also shown, that the massive enlargement of cells colonised with P. brassicae coincides with decreases in pectin methylation. In pme18-1 knock-out mutants P. brassicae could still induce demethylation; however, the galls in this line were smaller and cellular expansion was less pronounced. Alteration in pectin demethylation in the host resulted in changes in pathogen distribution and slowed down disease progression. To conclude, P. brassicae driven host organ hypertrophy observed during clubroot disease is accompanied by pectin demethylation in the extracellular matrix. The pathogen hijacks endogenous host mechanisms involved in cell wall loosening to create an optimal cellular environment for completion of its life cycle and eventual release of resting spores facilitated by degradation of demethylated pectin polymers.

Project description:Background: Histomonas meleagridis is an anaerobic, intercellular parasite, which infects gallinaceous birds such as turkeys and chickens. In recent years, the reemergence of Histomoniasis has caused serious economic losses as drugs to treat the disease have been banned. At present, research of H. meleagridis focuses on virulence, gene expression analysis, and the innate immunity of the host. However, there are no study on the differentially expressed miRNAs (DEMs) associated with the liver induced by H. meleagridis. In this research, high-throughput sequencing was used to analyze the expression profile of liver miRNA at 10 and 15 days post-infection (DPI) in chickens infected with Chinese JSYZ-F strain H. meleagridis. Results: Compared with the uninfected control, 120 and 118 DEMs were found in the liver of infected chickens at 10 DPI and 15 DPI, respectively, of which 74 DEMs were shared at two-time points. Differentially expressed miRNAs were classified into three types according to the time of infection: L1, 45 miRNAs differentially expressed only at 10 DPI, were predicted to target 1646 genes; L2, 43 miRNAs differentially expressed only at 15 DPI, were predicted to target 2257 genes; L3, 75 miRNAs differentially expressed at both 10 DPI and 15 DPI, were predicted to target 1623 genes. A total of 89, 87 and 41 significantly enriched GO terms (p<0.05) were identified at L1, L2 and L3, respectively. The KEGG pathway analysis of differentially expressed miRNA target genes were shown that 3, 4 and 5 significantly enriched KEGG pathway (p<0.05) were identified at L1, L2 and L3, respectively. Conclusion: This article suggested that liver miRNA expression was dynamically altered by H. meleagridis and host and showed that the expression pattern of the L1 class DEMs were more favourable for controlling the development of the inflammatory response, whereas the L2 class DEMs were more favourable for enhancing the inflammatory response. Inflammation-associated miRNA expression patterns were consistent with the liver inflammatory process after artificial infection. The results of the study laid the foundation for an in-depth analysis of the pathogenic mechanism of H. meleagridis from the perspective of host miRNAs.

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

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