Characterization of CRT1 family members in root-induced basal resistance, SAR, and ISR of Arabidopsis and barley
Final Report Abstract
Mounting evidence indicates that plant and animal MORC proteins play critical roles in gene silencing and disease progression. Our and others recent discovery that AtMORCs and HvMORCs interact with components of both the RdDM-pathway and the SWI/SNF chromatin remodeling complex provides a potential explanation for the silencing mechanism. Plant MORC proteins have been implicated in multiple layers of immunity. The discovery that AtMORC1 binds a wide variety of R proteins and the pattern recognition receptor (PRR) FLS2, and that the MORC-R protein interaction is disrupted by R protein activation, provides a small clue into how ETI and PTI are influenced by MORC proteins. However, many questions remain, including (i) how does disruption of the MORC-R protein interaction impact resistance signaling, (ii) what mechanism is responsible for disrupting this interaction, (iii) how do MORC proteins influence SAR and non-host resistance, and (iv) do MORCs have additional functions in the endosome? In addition, the mechanism through which MORC1 proteins from different species exert a positive or a negative influence on disease resistance remains unclear. Little was known about the role of MORC proteins of cereals, especially because KO mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. We used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III-dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. Corroborating and extending earlier findings, SpCas9- edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants. We also coraborated and extended ealier results that MORC proteins have a role in the regulation of plant defence responces and in epigenetic modifications but the underlying mechansms are still not fully understood. Yeast-Two-Hybrid system was used in combination with BiFC and subsequent verification steps such as western blots, to gain information on the possible interacting patterns of the MORC proteins in both Arabidopsis and barley and subcellular localization of the different homomeres and heteromers with the goal to gain an insight on the different signaling pathways that connect MORC proteins to both epigenetic regulations and defense mechanisms. Apart from scientific publications, our work resulted in some newspaper articles based on press releases from the JLU about the MORC project.
Publications
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(2014) The CRT1 subfamily of Microrchidia ATPases Regulates 1 Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens. Plant Physiology 164:866-878
Langen G, von Einem S, Koch A, Imani J, Pai S, Manohar M, Ehlers K, Choi HW, Claar M, Schmidt R, Mang HG, Bordiya Y, Kang HG, Klessig DF, Kogel KH
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(2015) Conserved nematode signaling molecules elicit plant defenses and pathogen resistance. Nature Comm 6:7795
Manosalva P, Manohar M, von Reuss S, Chen S, Koch A, Kaplan F, Choe A, Micikas R, Wang X, Kogel KH, Sternberg P, Williamson V, Schroeder F, Klessig D
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(2015) The GHKL ATPase MORC1 modulates species-specific plant immunity in Solanaceae. MPMI
Manosalva P, Manohar M, Kogel KH, Kang HG, Klessig F
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(2017) MORC proteins: novel players in plant and animal health. Front. Plant Sci.
Koch A, Kang HG, Steinbrenner J, Dempsey DA, Klessig DF, Kogel KH
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(2018) Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene editing system. Plant Biotechnol J.
Kumar N, Galli M, Ordon J, Stuttmann J, Kogel KH, Imani J
