Abstract
Type-III effector proteins are major virulence determinants that most gram-negative bacteria inject into host cells to manipulate cellular processes for infection. Because effector-targeted cells are embedded and underrepresented in infected plant tissues, it is technically challenging to isolate them for focused studies of effector-induced cellular changes. This protocol describes a novel technique, effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), for isolating biotin-labeled nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors by using streptavidin-coated magnetic beads. This protocol is an extension of the existing Nature Protocols Protocol of the INTACT method for the affinity-based purification of nuclei of specific cell types in the context of developmental biology. In a phytopathology scenario, our protocol addresses how to obtain eINTACT transgenic lines and compatible bacterial mutants, verify the eINTACT system and purify nuclei of bacterial effector-recipient cells from infected tissues. Differential analyses of purified nuclei from plants infected by bacteria expressing the effector of interest and those from plants infected by effector-deletion bacterial mutants will reveal the effector-dependent nuclear changes in targeted host cells. Provided that the eINTACT system is available, the infection experiment takes 5 d, and the procedures, from collecting bacteria-infected leaves to obtaining nuclei of effector-targeted cells, can be completed in 4 h. eINTACT is a unique method for isolating high-quality nuclei from bacterial effector-targeted host cells in native infection contexts. This method is adaptable to study the functions of type-III effectors from numerous gram-negative bacteria in host plants that are amenable to transformation.
Key points
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This protocol describes a method for purifying nuclei from live plant cells that have been specifically targeted by bacterial effectors during bacterial infection.
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Compared with similar affinity-based nuclei-isolation techniques that isolate cell type-specific nuclei such as INTACT, this method specifically isolates nuclei from host cells that have received bacterial effectors, allowing the interrogation of the effect of bacterial effectors on these cells in live infection models.
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Data availability
All data supporting this protocol are available in the main text or supplementary materials or are accessible via the original research article12. Source data are provided with this paper.
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Acknowledgements
We thank T. Lahaye (Eberhard-Karls-University Tübingen, Germany) for supplying the pDS300F and pDSK plasmids and a preliminary idea of a TALE-inducible INTACT system, L. D. Noël (Université de Toulouse, France) for contributing the Xcc bacterial strains and A.-G. Andrade-Galan for technical assistance. Our research benefits from the infrastructure at the ZMBP at the Eberhard-Karls-University Tübingen. This work was supported by the Institutional Strategy Program of the University of Tübingen (DFG, ZUK 63) and the German Research Foundation (DFG, no. 427105396) (both to Y.Y).
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Contributions
Y.Y. acquired funding, supervised the research and performed most experiments; Z.J. performed western blotting and microscopic analysis. Y.Y. analyzed the overall results and wrote the manuscript with input from Z.J.
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Nature Protocols thanks Adam Bogdanove, Roger Deal and Jordi Moreno-Romero for their contribution to the peer review of this work.
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Related links
Key reference using this protocol
You, Y. et al. Nat. Plants 9, 128–141 (2023): https://doi.org/10.1038/s41477-022-01302-y
This protocol is an extension to: Nat. Protoc. 6, 56–68 (2011): https://doi.org/10.1038/nprot.2010.175 and Nat. Protoc. 12, 238–254 (2017): https://doi.org/10.1038/nprot.2016.167
Extended data
Extended Data Fig. 1 XopD suppresses wilting and necrotic symptoms caused by Xcc∆xopD* infection.
Representative Arabidopsis leaves infected with Xcc∆xopD*AvrBs3 (−XopD) and Xcc*AvrBs3 (+XopD) at 7 DPI.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2
Source data
Source Data Fig. 2
Statistical data for Fig. 2b. Bacterial population per cm2 of Xcc*-inoculated leaves of the wild-type Col-0 plants and Xcc*AvrBs3-inoculated leaves of the eINTACT transgenic line at 7 DPI.
Source Data Fig. 2
Uncropped original gel pictures for Fig. 2c,d. c, Semi-quantitative RT-PCR analysis of RedNTF expression in leaves of the Arabidopsis eINTACT transgenic line infected with Xcc*AvrBs3 and Xcc*vec at 5 DPI. The expression of TUB2 and the genomic DNA of RedNTF and TUB loci (gRedNTF and gTUB) in the eINTACT transgenic line are used as controls. d, Uncropped original western blots. Enrichment of the RedNTF protein in eINTACT-purified nuclei. RedNTF and H3 protein detection via western blotting of the total protein extracts from Xcc*AvrBs3-infected and Xcc*vec-infected infected leaves and eINTACT-purified nuclei (nuceINTACT) from Xcc*AvrBs3-infected leaves. The blotted membrane was divided into two at 25 kDa, according to size marks in the protein ladder. The upper part (25–180 kDa) was used for detecting biotinylated-RedNTF protein (42 kDa) by using streptavidin alkaline phosphatase. The lower part (10–25 kDa) was used for detecting H3 protein by using an anti-H3 antibody. The abundance of H3 protein serves as an internal control of the number of nuclei in each sample.
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You, Y., Jiang, Z. The eINTACT method for studying nuclear changes in host plant cells targeted by bacterial effectors in native infection contexts. Nat Protoc 18, 3173–3193 (2023). https://doi.org/10.1038/s41596-023-00879-8
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DOI: https://doi.org/10.1038/s41596-023-00879-8
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