Arabidopsis thaliana CRT1 (compromised for recognition of Turnip Crinkle Virus) was previously shown to be required for effector-triggered immunity. Sequence analyses previously revealed that CRT1 contains the ATPase and S5 domains characteristic of Microchidia (MORC) proteins; these proteins are associated with DNA modification and repair. Here we show that CRT1 and its closest homologue, CRH1, are also required for pathogen-associated molecular pattern (PAMP)-triggered immunity, basal resistance, nonhost resistance and systemic acquired resistance. Consistent with its role in PAMP-triggered immunity, CRT1 interacted with the PAMP recognition receptor FLS2. Subcellular fractionation and transmission electron microscopy detected a subpopulation of CRT1 in the nucleus, whose levels increased following PAMP treatment or infection with an avirulent pathogen. These results, combined with the demonstration that CRT1 binds DNA, exhibits endonuclease activity, and affects tolerance to the DNA-damaging agent mitomycin C, argue that this prototypic eukaryotic member of the MORC superfamily has important nuclear functions during immune response activation.

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.

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Pro‐forms of growth factors have received increasing attention since it was shown that they can affect both the maturation and functions of mature growth factors. Here, we assessed the biological function of the pro‐form of bone morphogenetic protein‐2 (BMP‐2), a member of the transforming growth factor β (TGFβ)/ΒΜP superfamily. The role of the 263 amino acids of the pro‐peptide is currently unclear. In order to obtain an insight into the function of the pro‐form (proBMP‐2), the ability of proBMP‐2 to induce alkaline phosphatase (AP), a marker enzyme for cells differentiating into osteoblasts, was tested. Interestingly, in contrast to mature BMP‐2, proBMP‐2 did not lead to induction of AP. Instead, proBMP‐2 inhibited the induction of AP by BMP‐2. This result raised the question of whether proBMP‐2 may compete with mature BMP‐2 for receptor binding. ProBMP‐2 was found to bind to the purified extracellular ligand binding domain (ECD) of BMPR‐IA, a high‐affinity receptor for mature BMP‐2, with a similar affinity as mature BMP‐2. Binding of proBMP‐2 to BMPR‐IA was confirmed in cell culture by cross‐linking proBMP‐2 to BMPR‐IA presented on the cell surface. In contrast to this finding, proBMP‐2 did not bind to the ECD of BMPR‐II. ProBMP‐2 also differed from BMP‐2 in its capacity to induce p38 and Smad phosphorylation. The data presented here suggest that the pro‐domain of BMP‐2 can alter the signalling properties of the growth factor by modulating the ability of the mature part to interact with the receptors. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261817:BMPR‐IA (uniprotkb:http://www.ebi.uniprot.org/entry/P36894) and proBMP2 (uniprotkb:http://www.ebi.uniprot.org/entry/P12643) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by cross‐linking studies (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0030)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261681, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261693: BMP2 (uniprotkb:http://www.ebi.uniprot.org/entry/P12643) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to BMPR‐IA (uniprotkb:http://www.ebi.uniprot.org/entry/P36894) by enzyme linked immunosorbent assay (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0411)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261751, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261794: proBMP2 (uniprotkb:http://www.ebi.uniprot.org/entry/P12643) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to BMPR‐IA (uniprotkb:http://www.ebi.uniprot.org/entry/P36894) by competition binding (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0405)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261806, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7261846: BMPR‐IA (uniprotkb:http://www.ebi.uniprot.org/entry/P36894) physically interacts (http://www.ebi.ac.uk/ontology...

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The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutationcaused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire Nterminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.

Pro-forms of growth factors receive increasing attention since it was shown that they can affect the function(s) of the mature proteins. Here, we extend our previous investigations on the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2). We demonstrate that proBMP-2, upon prolonged incubation with C2C12 cells, induces alkaline phosphatase, a marker enzyme for osteoblastic differentiation. Expression studies with three different bone marker transcripts reveal that proBMP-2 induces bone-specific transcripts, however, to a smaller extent than the mature growth factor. To resolve this finding at the protein level, the fate of proBMP-2 and BMP-2 was studied in cell culture. We demonstrate that both proteins become internalized, and proBMP-2 is processed to mature BMP-2 within the cells. The data presented here suggest that proBMP-2 elicits biological functions as mature BMP-2 at a delayed and reduced level, which might depend on intracellular cleavage and subsequent secretion as mature BMP-2.

No abstract