The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

Abstract

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn 2+ -dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

Further analysis of barley MORC1 using a highly efficient RNA‐guided Cas9 gene‐editing system

“…Mutant lines developed less fungal colonies 6 days postinoculation (dpi) compared to wt plants ( hvmorc1 ‐L3: 71.5%; ‐L13: 71.8%; hvmorc1 ‐L16: 76%; Figure a). These results were consistent with our expectation that barley MORC paralogs respond similar to Bgh (Langen et al ., ). T1 plants from hvmorc1 ‐L10 and hvmorc1 ‐L13 that were homozygous for frameshift mutations ( hvmorc1‐1 and hvmorc1‐4 ; Figure S5) in the 5′ region of HvMORC1 were propagated for analyses of T2 plants.…”

“…Some 99.6% of retrotransposons are long terminal repeat (LTR) transposons, while 0.31% are non‐LTR retrotransposons (International Barley Genome Sequencing Consortium (IBSC), ). Notably, RNAi‐mediated KD of HvMORC1 did not result in detectable de‐repression of barley TEs (Langen et al ., ), suggesting that the remaining MORC protein activity (degree of gene KD was approx. 50%) was sufficient to repress TEs, which can explain the different phenotypes of RNAi‐generated hvmorc1 ‐KD vs. Sp Cas9‐mediated hvmorc1 ‐KO plants.…”

“…Seedlings were transferred to soil (Fruhstorfer Erde Typ T) and cultivated in a growth chamber at 22 °C/18 °C (day/night cycle) with 60% relative humidity and a photoperiod of 16 h (240 μmol/m 2 /s photon flux density). After complete emergence (12–14 day), the second leaves were detached, laid on 0.5% (w/v) water agar and inoculated with Bgh A6 (Langen et al ., ) at a density of 2 to 5 conidia mm −2 . For expression analysis, a high density of 10 to 15 conidia mm −2 was used.…”

“…Unlike Arabidopsis atmorc mutants, barley became more resistant to fungal pathogens, such as powdery mildew fungus Blumeria graminis f. sp. hordei ( Bgh ), when HvMORC2 , a paralog of HvMORC1 , was partially silenced by expressing MORC2 ‐targeting silencing constructs with inverted promoters in transgenic plants (Langen et al ., ). Consistent with this, transient overexpression of either of the five at that time‐known HvMORC paralogs compromised resistance to Bgh .…”

Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers

“…1073/pnas.1406611111/-/DCSupplemental. CRH6; Defective in Meristem Silencing 11 (DMS11)], and AtMORC7 (NP_194227; AT4G24970; CRH3) (25,(29)(30)(31)(32). AtMORC1 and AtMORC2 are the most closely related homologs and share 80.9% amino acid sequence identity (29)(30)(31)(32) (Fig.…”