Microtubule-organization during fungal invasion into barley epidermal cells
Final Report Abstract
Plant diseases and resistance to fungal pathogens are often considered as two sides of the same coin. In this sense, disease is the result of the failure of resistance due to a lack of pathogen recognition or suppression of host defenses or both. However, in case of long-lasting biotrophic interactions, it may be insufficient for the pathogen to just avoid recognition and suppress defense. Instead, a successful biotroph may take advantage of host susceptibility factors, which actively support the pathogen. The barley ROP (RHO OF PLANTs) GTPase RACB is a susceptibility factor during the interaction with the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh). In healthy plants, RACB is involved in cell polarity and cytoskeleton organization and we hypothesize that Bgh profits from these functions during accommodation of its haustorium. Indeed, a non-conventional effector of Bgh (ROP-interactive peptide 1) directly interacts with RACB and destabilizes microtubules. Therefore, and because model plant ROPs are involved in MT organization, we aimed at understanding the role of predicted MT-organizing ROP interactors of barley. RIC (ROP-Interactive CRIB Motif– Containing Proteins) and RIP/ICR (ROP Interactive Partner/Interactor of Constitutive Active ROPs) proteins were identified by bioinformatics means and selected based on their expression in barley leaves and their similarity to model plant proteins acting in MT organization. This put the focus mainly on barley RIPa, RIPb and RIC157. For all three proteins, we found evidence for direct protein-protein interaction with barley RACB, preferentially with the signalling-active GTP-bound version. This was shown in yeast and in planta. RIPa had a strong influence on MT-arrays in epidermal cells when co-expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1 and additionally was organized in MT-restricted plasma membrane domains. Our data support that ROP activity and MT organization is spatially organized in a mutualistic feedback between the activated ROP at the membrane and negative control of ROP activity from MTs by the MTlocalized ROP-regulator MAGAP1. This substantially adds to our understanding of ROP function in asymmetric plasma membrane and MT organization in plants, which is key to cell polarity. Further detailed analyses of protein functions and structure-function analyses identified the different RACB-interacting protein domains of RIPb and RIC157, and a strong and partially RACB-dependent susceptibility supporting effect of those two proteins, when overexpressed. The direct effects of these two proteins on MT organization appear weak, although at least RIPb is directly associated with MTs. The RACB-interacting coiled coil domain of RIPb is sufficient to support fungal invasion of barley cells and might be the executer domain of the protein or link to downstream executers. In this context, it is interesting that RIPb interacts in planta with a C2-domain protein, which is predicted to interact with phospholipids in a Ca2+-dependent manner and again supports fungal invasion into barley epidermal cells. Our results provide substantial insight in general ROP signalling in plants by identifying and analysing directly and indirectly RACB-interacting proteins in cereal crop plants, in which little is known about this important plant signalling branch. Our findings further pave the way for future understanding the mechanistic details of RIC and RIP protein function in disease susceptibility.
Publications
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(2019) Barley RIC157 is involved in RACB-mediated susceptibility to powdery mildew
Engelhardt S, Kopischke M, Hofer J, Probst K, McCollum C, Hückelhoven R
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(2018) A Barley powdery mildew fungus non-autonomous retrotransposon encodes a peptide that supports penetration success on barley. J. Exp. Bot. 69:3745-3758
Nottensteiner M, Zechmann B, McCollum C, Hückelhoven R
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(2018) Good riddance? Breaking disease susceptibility in the era of new breeding technologies. Agronomy 2018, 8(7), 114
Engelhardt S, Stam R, Hückelhoven R
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(2019) Barley ROP-interactive partner-a organizes into RAC1- and Microtubule-Associated ROP-Gtpase Activating Protein 1-dependent membrane domains
Hoefle, C, McCollum C, Hückelhoven R
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(2019) ROP Interactive Partner b interacts with the ROP GTPase RACB and supports fungal penetration into barley epidermal cells
McCollum C, Engelhardt S, Hückelhoven R
