ROP INTERACTIVE PARTNER b interacts with the ROP GTPase RACB and supports fungal penetration into barley epidermal cells

RHO of Plants (ROP) G-proteins are key components of cell polarization processes in plant development. The barley (Hordeum vulgare) ROP protein RACB, is a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). RACB also drives polar cell development, and this function might be coopted during formation of fungal haustoria in epidermal cells of barley. In order to understand RACB signaling during the interaction of barley with Bgh, we searched for potential downstream interactors of RACB. Here, we show that ROP INTERACTIVE PARTNER b (RIPb) directly interacts with RACB in yeast and in planta. Over-expression of RIPb supports susceptibility of barley to Bgh. RIPb further interacts with itself at microtubules. However, the interaction with activated RACB takes place at the plasma membrane. Both, RIPb and RACB are recruited to the site of fungal attack around the neck of developing haustoria suggesting locally enhanced ROP activity. We further assigned different functions to different domains of the RIPb protein. The N-terminal coiled-coil CC1 domain is required for microtubule localization, while the C-terminal coiled-coil CC2 domain is sufficient to interact with RACB and to fulfill a function in susceptibility at the plasma membrane. Hence, RIPb appears to locate at microtubules and is then recruited by activated RACB for a function at the plasma membrane during formation of the haustorial complex.

). It appears that in several monocots the first glutamic acid in the 151 QEEL motif is exchanged to aspartic acid (QDEL). We named these proteins 152 RIPa (HORVU3Hr1G087430), RIPb (HORVU1Hr1G012460) and RIPc 153 (HORVU3Hr1G072880), since we did not observe a clear orthology to 154 individual Arabidopsis RIP proteins and phylogenetic analysis was 155 ambiguous as well (Fig. 1B). We also identified three RIP proteins in rice 156 containing the QDEL motif as well as the QWRKAA motif (Os01g61760, RIP3/MIDD1 (At3g53350), RIP4 (At1g78430) and RIP5 (At5g60210)) show 161 little overall amino acid sequence conservation between the grasses and 162 Arabidopsis, except for the conserved QD/EEL motif at the N-terminus and 163 the QWRKAA motif at the C-terminus. The latter was shown to be necessary 164 for ROP interaction (Lavy et al. 2007). The alignment also shows 165 conservation of lysine residues at the very C-termini, which were shown 166 before to be important for membrane localization of other RIP proteins (Li 167 et al. 2008) (Fig. 1A). 168 Phylogenetic analysis shows that HvRIPa and HvRIPb are more closely 169 related to each other, than HvRIPc, which is located on an independent 170 branch of the tree (Fig. 1B). Two RIPs from rice (Oryza sativa ssp. japonica) 171 and two RIPs from Brachypodium distachyon (BRADI_2g54177v3,   representing membrane domains around papilla protrusions (Fig. 7B). Since

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RIPbCC2 had a stronger influence on fungal penetration success than full 304 length RIPb, we also imaged YFP-RIPbCC2 when co-expressed with CFP-305 CA RACB. Interestingly, there was a very strong co-localization of both 306 proteins around the haustorial neck region in penetrated cells, but also in 307 some instances at sites of repelled fungal attempts (Fig. 7C). The ring-like 308 accumulation of RIPbCC2 around the haustorial neck was also visible at 309 later stages of the interaction at 48 hours after the inoculation (Fig. 7D).

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There was also constantly local aggregation of cytoplasm at the sites of

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Our results support this, since only full length RIPb and truncations 324 containing this motif interacted with RACB and were subcellularly recruited 325 by CA RACB. (Fig. 4, Fig. 5, Supplemantal Fig. S2) vulgare. All identified RIPs from these four species also contain a conserved 328 QD/EEL motif located in an N-terminal CC1 domain (Fig. 1). The function of this motif, however, remains more elusive. Although the CC1 domain is 330 important for microtubule localization of RIPb (Fig. 5), amino acid 331 exchanges in the QDEL motif did not result a loss of microtubule association 332 (Supplemantal Fig. S3).

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Phylogenetic analyses show that both rice and Brachypodium, possess 334 putative orthologs of each of the three barley RIPs, implying possible 335 conserved function of the RIPs in grasses (Fig. 1) (Fig. 2, Supplemental Fig. S4A). Together with the fact that the RIPb  RIPb shows diverse subcellular localizations. Next to cytosolic localization, 359 we observed localization at the cell periphery and at the microtubule 360 cytoskeleton (Fig. 3). The N-terminal CC1 domain seems to be necessary does not interact with RACB (Fig. 2C, Fig. 5C)  a CaMV35S promotor was used. (Schweizer et al. 1999