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Cross-kingdom microRNA transfer for the control of the anthracnose disease in cassava

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Abstract

Cassava anthracnose disease (CAD), caused by Colletotrichum gloeosporioides f. sp. manihotis, is one of the most important diseases that cause significant yield loss in cassava. Recently, involvement of microRNAs (miRNAs), a class of small, single-stranded, non-coding RNAs, in the resistance against anthracnose disease has been indicated. In this study, two cassava cultivars that have different degree of CAD susceptibility were utilized to investigate the differences in defense responses these cultivars exhibited and to examine the role of seven miRNA families (mes-MIR156, 159, 164, 171, 396, 408, and 530) during CAD infection. Unlike the susceptible cultivar, the tolerant cultivar responded to fungal attack in the forms of hypersensitive response at the primary site of infection (or stem), as well as systemic induction of different defensive measures in the distal organs (or leaves) such as callose deposition, H2O2 accumulation, and upregulated expression of the miRNAs being studied. Two of the miRNAs, mes-MIR156 and mes-MIR164, were able to move across the kingdom boundary to the invading fungal cells. With the availability of genome sequence of C. gloeosporioides strain Cg-14, the mes-MIR156 and mes-MIR164 were predicted to target five and eleven fungal genes, respectively. Based on the differences in defense responses observed in the CAD-tolerant and CAD-susceptible cultivars, we then propose that the tolerant cultivar possesses a distinct defense mechanism against C. gloeosporioides f. sp. manihotis infection. In this defense mechanism, certain miRNAs are needed to help protect the host plant from the invading fungal pathogen.

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Data availability

The dataset generated during and/or analyzed during the current study are not publicly available due to the data ownership policy of the institutions the authors belong but are available from the corresponding author on reasonable request.

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Acknowledgements

This research work was supported by the Center of Excellence on Agricultural Biotechnology, Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (AG-BIO/MHESI). A scholarship from the Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency, Thailand, was gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Nattaya Pinweha, Punchapat Sojikul, Pakatorn Sae-Lim & Jarunya Narangajavana

  2. Center of Excellence on Agricultural Biotechnology (AG-BIO/MHESI), Bangkok, 10900, Thailand

    Nattaya Pinweha & Jarunya Narangajavana

  3. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, 12120, Thailand

    Supatcharee Netrphan

  4. Department of Plant Science, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Unchera Viboonjun

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  1. Nattaya Pinweha
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Contributions

JN conceived and designed experiments. NP conducted experiments. P. Sae-Lim analyzed the results. SN, JN, P. Sojikul, and UV discussed the results. SN wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Supatcharee Netrphan.

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Supplementary Information

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40858_2022_503_MOESM1_ESM.doc

Supplementary file1 (DOC 569 KB) Recovery of fungal hyphae from the stems of CAD-infected HB60 and HN. After 4 days of the introduction of C. gloeosporioides f. sp. manihotis spore suspension to HB60 and HN planlets through the puncture wound (black arrow), the stems of CAD-infected plants were cut into pieces and labeled A to E depending on their location either above (A, B and C) or below (D, E and F) the infected point (A). The cut pieces of stems were then placed on PDA plates and incubated at 30°C for 5 days (B). Fungal growth was observed only from the stems of plants infected with C. gloeosporioides f. sp. manihotis but not from those exposed to sterile water (control).

Supplementary file2 (DOC 64 KB) List of primers used in this study

40858_2022_503_MOESM3_ESM.doc

Supplementary file3 (DOC 216 KB) Specificity of the primers used in qRT-PCR. Each experiment was performed in triplicate. Based on the melting or dissociation curves, amplification of the genes of interest with selected primer pairs yielded only a single amplicon.

40858_2022_503_MOESM4_ESM.doc

Supplementary file4 (DOC 398 KB) Schematic maps of putative cis-acting elements identified by in silico analysis of the 3-kb upstream sequence of seven miRNA gene families and their target genes in cassava. Positions are with respect to the first base of the translation start site (ATG).

40858_2022_503_MOESM5_ESM.xlsx

Supplementary file5 (XLSX 16 KB) Radial growth of C. gloeosporioides f. sp. manihotis on PDA plates spread with various types of cassava phloem exudates

40858_2022_503_MOESM6_ESM.doc

Supplementary file6 (DOC 62 KB) Mapping of mes-MIR156 and mes-MIR164 with predicted target transcripts of C. gloeosporioides Cg-14 (GCA_000446055). The mes-MIR156 and mes-MIR164-guided cleavage sites were predicted after the 10th position of the miRNA 5´ end (arrows). Watson-Crick pairing (:) and wobble pairing (.) are indicated.

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Pinweha, N., Netrphan, S., Sojikul, P. et al. Cross-kingdom microRNA transfer for the control of the anthracnose disease in cassava. Trop. plant pathol. 47, 362–377 (2022). https://doi.org/10.1007/s40858-022-00503-2

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