Magnaporthe oryzae CK2 is involved in rice blast pathogenesis and accumulates in nuclei, nucleoli, at septal and appressoria pores and forms a large ring structure in appressoria

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures as septa and appressorial pores.

regulatory subunit. In contrast, localization near septa, required all three subunits. 23 The ~1300 proteins co-immunoprecipitating with the catalytic subunit were highly 24 enriched for those known to reside at these locations. Appressoria contain a 25 filamentous form of CK2. The interacting proteins in hyphae were enriched for 26 intrinsically disordered proteins with characteristics previously identified as being a 27 mechanism for regulation of protein aggregation. Examining gene expression profiles, 28 we find a correlation of CK2 expression with genes for protein disaggregation and 29 autophagy. Our observations support the view that CK2 plays a key role in Introduction 34 Since its discovery (Meggio and Pinna, 2003), the constitutive serine/threonine (S/T) 35 kinase activity of CK2 and the increasing number of proteins it has been shown to 36 phosphorylate have puzzled scientists (Ahmad et al., 2008;Götz and Montenarh, 37 2016; Meggio and Pinna, 2003). Indeed CK2 has been implicated in a wide range of 38 cellular processes (Götz and Montenarh, 2016). The CK2 holoenzyme is a 39 heterotetrameric structure consisting of 2 catalytic α-units and 2 regulatory β-40 subunits (Ahmad et al., 2008). In mammals, there exist two different alpha subunits α 41 (a1) and α' (a2) and the enzyme can contain any combination of α-subunits 42 (α1α1, α1α2, α2α2) combined with the β-subunits. In Saccharomyces cerevisiae, 43 CK2 contains two different alpha-and two different β-subunits (b1 and b2) 44 (Padmanabha et al., 1990). CK2 has been extensively studied in the budding yeast S. 45 cerevisiae (Padmanabha et al., 1990), however, functions of CK2 involved in 46 multicellularity might be obscured in yeast. For fungi, it has been reported that 47 We attempted targeted deletions of the three identified genes and succeeded in 80 deleting the two CKb genes but not the CKa and then also saw that the conidial 81 morphology was different in the CKb mutants in that they had fewer conidial 82 compartments ( Figure 2). ΔMockb1 and ΔMockb2 deletion mutants, thus, we used mycelial plugs to test for 119 infection (Liu et al., 2010;Talbot et al., 1996). Compared to the background strain  The localization pattern suggested that CK2 may have substrates associated with 144 septa and nucleolar function. To explore this, we performed co-immunoprecipitation 145 to identify proteins interacting with CK2 using GFP-CKa as a "bait", and in addition 146 to the bait, identified 1505 proteins (Supplementary File 1). We also searched the M. Fisher's Exact test) lending support for the proposed role for this motif as a target for 158 CK2 phosphorylation and protein unfolding. As expected, the pulldown caught both 159 CKb proteins.

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Since CK2 localizes to septa we looked for known septal proteins in the pulldown.

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All previously identified proteins by Dagas et al. (Dagdas et al., 2012)  with each other to form ordered subregions that have been described as 185 membraneless organelles, such as nucleoli (Wright and Dyson, 2015). Since CK2 186 localizes to the nucleolus we were especially interested in the interaction of CK2 with 187 nucleolar localized proteins. We identified homologues to the well described S.  There was no enrichment for specific pathogenicity related proteins (Funcat category 204 32.05 disease, virulence and defence) (Supplementary File 4). This is generally true 205 within the whole Funcat category related to stress and defence (32 CELL RESCUE, 206 DEFENSE AND VIRULENCE) with the exception of proteins involved in the 207 unfolded protein response (32.01.07 unfolded protein response) (e.g. ER quality 208 control), which were overrepresented. This is notable since an involvement of CK2 in 209 protein import into the ER has be established (Wang and Johnsson, 2005). An  Since CK2 activity has the potential to favour protein-protein binding between 227 intrinsically disordered proteins it consequently also has the potential to enhance 228 protein aggregation. Some of these unfolded proteins may trigger the unfolded 229 protein response involved in disaggregation. Hsp104 is a disaggregase that cooperates    The crystal structure suggested that CK2 can form filaments and higher-order 287 interactions between CK2 holoenzyme tetramer units, and based on this it has been 288 predicted that autophosphorylation between the units could occur to down-regulate 289 activity (Litchfield, 2003;Poole et al., 2005). Filament formation has been shown to 290 occur in vitro (Glover, 1986;Seetoh et al., 2016;Valero et al., 1995) and in vivo 291 (Hübner et al., 2014). Several forms of higher order interactions have been predicted, 292 and it has been demonstrated that at least one of these has reduced kinase activity 293 (Poole et al., 2005;Valero et al., 1995). However, in our localization experiments we 294 cannot distinguish if the large structure is due to co-localization of the CK2 with

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Previous studies of subcytosolic localization reveals that this enzyme is also 320 associated with import into organelles. CK2 promotes protein import into  Since these proteins need to be imported into mitochondria in an unfolded state, this 331 may point to the existence of other CKa phosphorylation and unfolding motifs that 332 help keep these proteins unfolded until they reach their destination inside the 333 mitochondria.

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To have such dynamic function as an unfolder of proteins by phosphorylation, CK2 335 should be partnered with phosphatases as counterparts and their activity may track 336 CK2 activity. Consistent with this possibility, we found that two of the five S/T 337 phosphatases that are present in the pulldown are strongly co-regulated with CKa 338 (Figure 9), further supporting the view that CKa-dependent 339 phosphorylation/dephosphorylation plays a major role in shaping protein interactions.

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Together with the high expression of CK2 in cells, this suggests an important 341 function of CK2 as a general temporary unfolder of intrinsically disordered proteins 342 that comprise roughly 30 % of eukaryotic proteins (Vucetic et al., 2003). 343 As MoCK2 is present in the cytoplasm and nucleoplasm it could generally assist 344 intrinsically disordered proteins forming larger protein complexes (Uversky, 2015). It 345 also seems to be essential for assembling ribosomes containing large numbers of 346 intrinsically disordered proteins (Uversky, 2015). All these functions also explains 347 why CK2 is needed constitutively (Meggio and Pinna, 2003).

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In the absence of well-functioning autophagy removing incorrectly formed larger 349 protein aggregates, like those formed in brain cells of Alzheimer's patients (Zare-    Table   407 2. This fragment was inserted into the pCB1532 to construct the complementation  As for the ΔMoCKb1 deletion mutant, we constructed a knockout vector to delete the 413 MoCKb2 from the background strain Ku80. All the primers are listed in the Table 2.  The construction of localization vectors 429 In order to detect the localization of MoCK2, we constructed localization vectors.

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The vector pCB-3696OE containing the RP27 strong promoter was used to detect the

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Estimation of non-specific binding of proteins in the pulldown 475 We developed two methods to estimate the number of non-specific binding proteins 476 found in the CKa pulldown. The first approach is a chemistry-based reasoning and 477 assumes that the degree of unspecific association to the protein per protein surface 478 area is the same for GFP specific hits and for the CK2 holoenzyme pulled down.  The data that support the findings of this study are available from the corresponding 525 authors upon reasonable request.                 Phosphatase MGG_01690T0 Figure 10 Plot of expression involved in protein quality control vs MoCKa expression in a range of transcriptomes from different experiments (Note: Log2 scale on axes and grids are represented with fixed "aspect ratio" to highlight the slope of the correlation). P value for the Null hypothesis that there is no correlation = 9.9E-10. Transcriptomic data was downloaded from public websites so as to be able to test the relationship under many different growth conditions in many experiments. Hsp104, SSa1 and Ymdj1 has in yeast been shown to cooperatively help aggregate proteins to be able to refold. The key protein with its main function in this process appear to be Hsp104 (Glover and Lindquist, 1998). Transcriptomic data was downloaded from public websites so as to be able to test the relationship under many different growth conditions in many experiments. Hsp104, SSa1 and Ymdj1 has in yeast been shown to cooperatively help aggregate proteins to be able to refold. The key protein with its main function in this process appear to be Hsp104 (Glover and Lindquist, 1998) . (Note: Log2 scale on axes and grids are represented with fixed "aspect ratio" to highlight the slope of the correlation). P value for the Null hypothesis that there is no correlation = 3.6E-08 . Transcriptomic data was downloaded from public websites so as to be able to test the relationship under many different growth conditions in many experiments. Log 2 ATG8 expression Log 2 CK2 expression