Barley RIC157 is involved in RACB-mediated susceptibility to powdery mildew

Abstract

9Successful pathogens often benefit from certain cellular host processes. For the 10 biotrophic ascomycete fungus Blumeria graminis f.sp. hordei (Bgh) it has been shown 11 that barley RACB, a small monomeric G-protein (ROP, RHO of plants), is required for 12 full susceptibility to fungal penetration. The susceptibility function of RACB probably 13 lies in its role in cell polarisation, which may be co-opted by the pathogen for invasive 14 ingrowth of its haustorium. However, the actual mechnism of how RACB supports the 15 fungal penetration success is little understood. RIC proteins are considered scaffold 16 proteins which can interact directly with ROPs via a conserved CRIB motif. Here we 17 describe a yet uncharacterised RIC protein, RIC157, which can interact directly with 18 RACB. We could show that RIC157 undergoes a recruitment from the cytoplasm to the 19 cell periphery in the presence of activated RACB. During fungal infection, RIC157 and 20 activated RACB colocalise at the penetration site, particularly at the haustorial neck. In 21 a RACB-dependent manner, transiently overexpressed RIC157 renders barley 22 epidermal cells more susceptible to fungal penetration. We conclude that RIC157 23 promotes fungal penetration into barley epidermal cells via its function as downstream 24 executor in RACB-signaling. 25 26 93 polarisation and cytoskeleton organisation, ROPs have been also implicated in 94 membrane trafficking and auxin signaling (Yalovsky et al. 2008, Wu et al. 2011).95 OsRac1 from rice (Oryza sativa), a great example for demonstrating the versatility of 96 ROPs, enhances cell division by regulating OsMAPK6, thereby promoting rice grain 97 yield (Zhang et al. 2019). It has also been shown to regulate immune-related processes 98 like ROS production, defense gene expression and cell death. OsRac1 becomes 99 activated by OsRacGEF1 upon receptor-mediated perception of fungal-derived chitin 100 4 by OsCEBiP and OsCERK1 (Akamatsu et al. 2013). Chitin-perception might also lead 101 to the activation of OsRAC1 by OsSWAP70 (Yamaguchi et al. 2012). Downstream 102 signaling by OsRAC1 is also triggered after recogniton of pathogen effector proteins: 103 Plasma membrane-localised Pit, a nucleotide binding-leucine rich repeat resistance 104 (NLR) protein for the rice blast fungus Magnaporte oryzae, associates with DOCK 105 family GEF OsSPK1, thereby likely activating OsRac1 (Kawano et al. 2010, Kawano 106 et al. 2014, Wang et al. 2018). A recent report regarding an involvement in defence 107 reactions against rice blast mediated by the NLR protein PID3 (Zhou et al. 2019) opens 108 up the possibility of OsRac1 being a downstream hub of other rice NLR proteins. 109 In the barley-powdery mildew interaction, several barley proteins involved in ROP 110 signaling or ROP activity regulation have been shown to influence fungal penetration 111 success. The barley ROP RACB has been shown to act as susceptibility factor 112 (Schultheiss et al. 2002, Schultheiss et al. 2003, Hoefle et al. 2011). In the absence of 113 t...

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

“…In both scenarios, RACB signaling would be effectively inhibited. In turn, it is likely that interaction of activated RACB with CRIB-domaincontaining executors, such as RIC proteins (Schultheiss et al, 2008;Engelhardt et al, 2019), would mask the ubiquitination site. This could not only lead to increased protein stability, but also secure RACB's signaling activity during crucial processes.…”

“…For the analysis of Bgh susceptibility after transient overexpression of untagged CA-RACB(-K167R), a full-length cDNA clone of RACB was first amplified with Gateway® attachment sites using primers RACB-Gateway-F + RACB-Gateway-R and cloned into the Gateway® entry vector pDONR223 using routine Gateway® BP-cloning procedures (Invitrogen, Carlsbad, California, USA). From pDONR223, RACB was transferred into the Gateway®-compatible plant expression vector pGY1-GW (generated in Engelhardt et al (2019)) using routine Gateway® LR-cloning. A CA-RACB-K167R clone was generated from pGY1-RACB using subsequent site-directed mutagenesis with primers RACB-SDM-G15V-F + RACB-SDM-G15V-R and RACB-SDM-K167R-F + RACB-SDM-K167R-R.…”

“…Among these barley ROPs, RACB is the best characterized ROP family member. Under physiological conditions, it is required for root hair outgrowth and stomatal subsidiary cell formation, and RACB and its interacting proteins further steer cytoskeleton organization (Engelhardt et al, 2019). During Bgh attack, RNA-interference (RNAi)-mediated knockdown of RACB decreased the penetration success of the fungus into barley epidermal cells, whereas overexpression of a constitutively activated RACB variant (CA, RACB-G15V, a GTPase mutant that cannot hydrolyse GTP) promoted susceptibility (Schultheiss et al, 2002;Schultheiss et al, 2003;Pathuri et al, 2008;Hoefle et al, 2011).…”

“…Since RHO family proteins interact with several downstream executors to perform their signaling function, interaction partners of RACB were investigated with regard to barley susceptibility to Bgh. It was shown that two classes of scaffolding proteins, Interactor of Constitutively Active ROPs/ROP-interactive partners (ICRs/RIPs) and ROP-interactive and CRIB-motif containing proteins (RICs), associate with CA-RACB at the plasma membrane and enhance penetration success of Bgh into barley epidermal cells when transiently overexpressed (Schultheiss et al, 2008;Engelhardt et al, 2019;McCollum et al, 2020). In contrast, two other interactors of activated RACB support resistance to fungal penetration.…”

Regulation and Functions of ROP GTPases in Plant–Microbe Interactions

“…HvRACB and HvRIC157 together localize at the cell periphery or plasma membrane where B. graminis f.sp. hordei invades susceptible barley cells [ 83 ]. Similarly, HvRACB and HvRIC171 form a protein complex at sites of fungal entry [ 84 ].…”