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Dynamic regulation of Pep-induced immunity through post-translational control of defence transcript splicing

Abstract

The survival of all living organisms requires the ability to detect attacks and swiftly counter them with protective immune responses. Despite considerable mechanistic advances, the interconnectivity of signalling modules often remains unclear. A newly characterized protein, IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), negatively regulates immune responses in both maize and Arabidopsis, with disrupted function resulting in enhanced disease resistance. IRR associates with and promotes canonical splicing of transcripts encoding defence signalling proteins, including the key negative regulator of pattern-recognition receptor signalling complexes, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). On immune activation by Plant Elicitor Peptides (Peps), IRR is dephosphorylated, disrupting interaction with CPK28 transcripts and resulting in the accumulation of an alternative splice variant encoding a truncated CPK28 protein with impaired kinase activity and diminished function as a negative regulator. We demonstrate a new mechanism linking Pep-induced post-translational modification of IRR with post-transcriptionally mediated attenuation of CPK28 function to dynamically amplify Pep signalling and immune output.

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Fig. 1: AtPep1 affects the phosphorylation of IRR in a time- and concentration-dependent manner.
Fig. 2: IRR mutants are hypersensitive to AtPep1 treatment.
Fig. 3: IRR is implicated in defence and alternative splicing.
Fig. 4: IRR affects the ratio of CPK28-RI splice variants and CPK28 function.
Fig. 5: Association of IRR with CPK28 transcript is phosphorylation-dependent.
Fig. 6: Proposed model by which IRR dynamically regulates CPK28 immunomodulatory buffering of PEPR-mediated immunity.

Data availability

The raw read sequences are deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE146282. The data generated and analysed in this study are included in the published article and Supplementary Information. All data are available from the corresponding author upon request.

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Acknowledgements

We thank S. A. Whitham (Iowa State University Plant Sciences Institute) for providing the constructs for the VIGS experiments, and A. Groisman (University of California San Diego Department of Physics) for the use of his Biolistic inoculation apparatus. This work was funded by NSF CAREER Award no. 1943591, a Hellman Foundation Fellowship and UC San Diego Start-up funds to A.H. K.D. was additionally funded by Ciências sem Fronteiras/CNPq fellowship no. 200260/2015‐4. E.P. was additionally funded by the Cell and Molecular Genetics (CMG) Training Program at the University of California, San Diego. Z.S. and S.P.B. were funded by NSF award no. 1546899.

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A.H. and K.D. conceived the project. K.D. conducted the experiments. A.H. and K.D. analysed the data and wrote the manuscript. P.R.W. performed the MAP kinase assay, confocal microscopy experiments and phylogenetic analysis. E.P. analysed the leaf volatile emissions and helped edit the figures. Y.T. performed the in-gel kinase activity assays. C.V. assisted with the generation of the transgenic plant lines. Z.S. and S.P.B. performed the phosphoproteomic analysis. J.I.S. contributed critical experimental resources.

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Correspondence to Alisa Huffaker.

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Supplementary Information

Supplementary Figs. 1–24, references and methods.

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Supplementary Tables

Supplementary Table 1: differentially expressed genes (DEG); Supplementary Table 2: upregulated genes in irr-1 compared with the wild type; Supplementary Table 3: alternative splicing analysis; Supplementary Table 4: list of primers used.

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Dressano, K., Weckwerth, P.R., Poretsky, E. et al. Dynamic regulation of Pep-induced immunity through post-translational control of defence transcript splicing. Nat. Plants 6, 1008–1019 (2020). https://doi.org/10.1038/s41477-020-0724-1

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