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Differential Expression of miRNAs in Brassica Napus Root Following Infection With Plasmodiophora Brassicae

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Differential Expression of miRNAs in Brassica Napus Root Following Infection With Plasmodiophora Brassicae

Shiv S Verma et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Morphology if 10-day old healthy (A), and clubroot-infected (B), and 20 day old uninfected (C), and clubroot infected (D) B. napus roots showing gall formation in the latter (open arrows) due to P. brassicae infection.
Histopathological analysis indicates the presence of evacuated zoosporangia (open arrow) in the root hair and primary plasmodia (closed arrows) in epidermal cells indicating infection (F) compared to uninoculated controls (E). At 20 dpi, the cortical cells of infected tissue show the presence of numerous secondary plasmodium (arrowheads) in the cortical cells (H) compared to the control (G). Bars represent 5 mm for Figures. A–D and 100 µm for Figures. E–H.
Figure 2
Figure 2. miRNA-microarray expression of P. brassicae responsive miRNAs exhibiting differential expression at 10- (A) and 20- dpi (B, C) following pathogen infection.
Figure 3
Figure 3. Relative abundance of miRNA in B. napus plant infected with P. brassicae.
(a) Relative accumulation of miRNA showing the quantitative expression at 10 dpi and (b) Relative accumulation of miRNA showing the quantitative expression at 20 dpi. The expression of sonR66 was used as internal control in the experiment. The error bars show the standard deviation.
Figure 4
Figure 4. miRNA mapping and cleavage site determination through 5′ RLM RACE.
Agarose gel image of 5′ RACE products (A) and the target mRNA cleavage sites (B). The targeted mRNA section and miRNA sequences, along with mismatch (es), if any, are shown as the expanded region. The 5′ends of the cleaved product determined by sequencing is indicated by the vertical arrowheads, along with the numbers of clones analyzed. The horizontal arrowheads indicated the gene-specific primer sites used for 5′RLM-RACE.

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Funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Agriculture Funding Consortium, Alberta Canola Producers Commission, and Alberta Crop Industry Development Fund is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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