Rice blast infection of Brachypodium distachyon as a model system to study dynamic host/pathogen interactions

Abstract

Interactions between plants and compatible fungal pathogens are spatially and temporally dynamic, posing a major challenge for sampling and data analysis. A protocol is described for the infection of the model grass species Brachypodium distachyon with Magnaporthe grisea (rice blast), together with modifications to extend the use to rice and barley. We outline a method for the preparation of long-term stocks of virulent fungal pathogens and for the generation of fungal inoculants for challenge of host plants. Host plant growth, pathogen inoculation and plant sampling protocols are presented together with methods for assessing the efficiency of both infection and sampling procedures. Included in the anticipated results is a description of the use of metabolite fingerprinting and multivariate data analysis to assess disease synchrony and validate system reproducibility between experiments. The design concepts will have value in any studies using biological systems that contain dynamic variance associated with large compositional changes in sample matrix over time.

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Figure 1: Viability testing of Magnaporthe grisea conidia by measuring germination and appressorium production in vitro.
Figure 2: Preparation of long-term stocks of virulent Magnaporthe grisea.
Figure 3: CMA plates of Magnaporthe grisea Guy11 cultures grown at 26 °C with a 16-h light and dark cycle for conidia harvesting.
Figure 4: Brachypodium distachyon ABR1 seedlings grown for 21–28 d in an environmentally controlled growth room under a 16-h light period at 23 °C (±2 °C).
Figure 5: Development of disease symptoms on Brachypodium distachyon ABR1 leaves after infection with Magnaporthe grisea Guy11.
Figure 6: Confirmation of pathogen from lesions and maintaining pathogenicity of Magnaporthe grisea stocks.
Figure 7: Brachypodium distachyon (ABR 1) at five-leaf stage indicating harvest leaf and sampling zone.
Figure 8: During disease progression, infected Brachypodium distachyon leaves become gradually more desiccated, altering the leaf wet weight but not biomass.
Figure 9

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Acknowledgements

We thank all collaborators who provided valuable samples to develop this protocol and in particular Rob Darby for maintaining laboratory infrastructure, equipment and materials. Metabolite analysis, statistical work and biological materials used in example data were generated as part of the UK Biotechnology and Biological Sciences Research Council grants BB/D006953/1 (D.P. and J.D.), BB/D006791/1 (N.T. and Z.C.R.).

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Correspondence to John Draper.

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Parker, D., Beckmann, M., Enot, D. et al. Rice blast infection of Brachypodium distachyon as a model system to study dynamic host/pathogen interactions. Nat Protoc 3, 435–445 (2008). https://doi.org/10.1038/nprot.2007.499

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