Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 30:15:1391900.
doi: 10.3389/fpls.2024.1391900. eCollection 2024.

The fungicide pyraclostrobin affects gene expression by altering the DNA methylation pattern in Magnaporthe oryzae

Shumei Fang  1   2 , Hanxin Wang  2 , Kaihua Qiu  1 , Yuanyuan Pang  2 , Chen Li  1 , Xilong Liang  1
Affiliations

The fungicide pyraclostrobin affects gene expression by altering the DNA methylation pattern in Magnaporthe oryzae

Shumei Fang et al. Front Plant Sci. .

Abstract

Introduction: Rice blast disease caused by Magnaporthe oryzae has long been the main cause of rice (Oryza sativa L.) yield reduction worldwide. The quinone external inhibitor pyraclostrobin is widely used as a fungicide to effectively control the spread of pathogenic fungi, including M. oryzae. However, M. oryzae can develop resistance through multiple levels of mutation, such as target protein cytb mutation G143A/S, leading to a decrease in the effectiveness of the biocide after a period of application. Therefore, uncovering the possible mutational mechanisms from multiple perspectives will further provide feasible targets for drug development.

Methods: In this work, we determined the gene expression changes in M. oryzae in response to pyraclostrobin stress and their relationship with DNA methylation by transcriptome and methylome.

Results: The results showed that under pyraclostrobin treatment, endoplasmic reticulum (ER)-associated and ubiquitin-mediated proteolysis were enhanced, suggesting that more aberrant proteins may be generated that need to be cleared. DNA replication and repair processes were inhibited. Glutathione metabolism was enhanced, while lipid metabolism was impaired. The number of alternative splicing events increased. These changes may be related to the elevated methylation levels of cytosine and adenine in gene bodies. Both hypermethylation and hypomethylation of differentially methylated genes (DMGs) mainly occurred in exons and promoters. Some DMGs and differentially expressed genes (DEGs) were annotated to the same pathways by GO and KEGG, including protein processing in the ER, ubiquitin-mediated proteolysis, RNA transport and glutathione metabolism, suggesting that pyraclostrobin may affect gene expression by altering the methylation patterns of cytosine and adenine.

Discussion: Our results revealed that 5mC and 6mA in the gene body are associated with gene expression and contribute to adversity adaptation in M. oryzae. This enriched the understanding for potential mechanism of quinone inhibitor resistance, which will facilitate the development of feasible strategies for maintaining the high efficacy of this kind of fungicide.

Keywords: Magnaporthe oryzae; adenine methylation; cytosine methylation; gene expression; pyraclostrobin.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic overview of the methyl-seq and RNA-seq protocol. Mycelia cultured after 24 h treatment (Final concentration of pyraclostrobin was 7.4 µg/L, and control was added the same volume of sterile water) were collected from each flask and were divided into two parts for DNA extraction and RNA extraction. After nanopore sequencing and Illumina sequencing, data analysis was performed to obtain differentially methylated genes and differentially expressed genes, and then possible metabolite process was analyzed by both GO and KEGG pathway.
Figure 2
Figure 2
Gene expression changes and differentially expressed genes (DEGs) involved in metabolic processes under pyraclostrobin stress. (A) Volcano plot of differentially expressed genes after pyraclostrobin treatment; (B) KEGG pathway analysis of differentially expressed genes. Among the 564 (DEGs) detected, 340 genes were upregulated, and 224 genes were downregulated. These genes were clustered into five main classes: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems.
Figure 3
Figure 3
Endoplasmic reticulum-associated and ubiquitin-mediated proteolysis. Red indicates upregulated proteins; green indicates downregulated proteins. Multiple molecular chaperones that can recognize and bind nascent peptides, proteins involved in the formation of ubiquitin ligase complexes, and proteins that facilitate the entry of ubiquitin-binding proteins into the proteasome were upregulated, but proteins involved in the correct folding of glycoproteins were downregulated.
Figure 4
Figure 4
Glutathione metabolism and generation of trypanothione and tryparedoxin. Glutathione S-transferase (GST), glutathione reductase (GSR) and glutathione peroxidase (GPX) were upregulated and contributed to the removal of hydrogen peroxide and other toxic substances. Ribonucleoside-diphosphate reductase M1 (RRM1) was downregulated, which contributed to high concentrations of trypanothione and tryparedoxin.
Figure 5
Figure 5
Alternative splicing after pyraclostrobin treatment. The MGG_00470, MGG_09435, MGG_11079, MGG_10183 and MGG_04006 genes exhibited exon inclusion. MGG_06981 exhibited an alternative 5′ splice site.
Figure 6
Figure 6
DNA methylation levels in gene regions and their changes after pyraclostrobin treatment. Pyr, pyraclostrobin; CK, control. TSS, transcriptional start site; TES, transcriptional end site. The m5C level was greater than the m6A level. At the transcriptional start site and transcriptional end site, the m5C level was high, while the m6A level was low.
Figure 7
Figure 7
Changes in the differential methylation of genes caused by pyraclostrobin. P_0-1k, promoter_0-1 kb; P_1-2k, promoter_1-2 kb; P_2-3k, promoter_2-3 kb; Hypo, hypomethylation; Hyper, hypermethylation.
Figure 8
Figure 8
GO and KEGG enrichment of differentially methylated genes (DMGs) in the pyraclostrobin group compared to the control group. (A) Top 20 enriched GO terms of 5mC DMGs. (B) Top 20 enriched KEGG pathways of 5mC DMGs. (C) Top 20 enriched GO terms of the 6mA DMGs. (D) Top 20 enriched KEGG pathways of the 6mA DMGs. m5C and m6A DMGs were mainly associated with RNA polymerase II transcription factor, transcription, ubiquitin protein ligase, proteolysis, and nucleotide excision repair and protein processing in the ER. These critical terms were highlighted by underlines in the figure.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Akalin A., Kormaksson M., Li S., Garrett-Bakelman F. E., Figueroa M. E., Melnick A., et al. . (2012). MethylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 13, R87. doi: 10.1186/gb-2012-13-10-r87 - DOI - PMC - PubMed
    1. Anders S., Huber W. (2010). Differential expression analysis for sequence count data. Nat. Preced 1–10. doi: 10.1038/npre.2010.4282.2 - DOI - PMC - PubMed
    1. Angermueller C., Clark S. J., Lee H. J., Macaulay I. C., Teng M. J., Hu T. X., et al. . (2016). Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity. Nat. Methods 13, 229–232. doi: 10.1038/nmeth.3728 - DOI - PMC - PubMed
    1. Anzmann A. F., Sniezek O. L., Pado A., Busa V., Vaz F. M., Kreimer S. D., et al. . (2021). Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin. BioRxiv, 2021-01. doi: 10.1101/2021.01.06.425502 - DOI
    1. Arechederra M., Daian F., Yim A., Bazai S. K., Richelme S., Dono R., et al. . (2018). Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer. Nat. Commun. 9, 3164. doi: 10.1038/s41467-018-05550-5 - DOI - PMC - PubMed

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Incubation Subjects of Heilongjiang Bayi Agricultural University [grant number XZR2014-01], the Provincial and Natural Science Foundation of Heilongjiang, China [grant number C2016047] and PhD research startup foundation of Heilongjiang Bayi Agricultural University [XDB-2016-01].

LinkOut - more resources

-