The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant’s Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA-induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility.

Rho proteins of plants (ROPs) form a specific clade of Rho GTPases, which are involved in either plant immunity or susceptibility to diseases. They are intensively studied in grass host plants, in which ROPs are signaling hubs downstream of both cell surface immune receptor kinases and intracellular nucleotide-binding leucine-rich repeat receptors, which activate major branches of plant immune signaling. Additionally, invasive fungal pathogens may co-opt the function of ROPs for manipulation of the cytoskeleton, cell invasion and host cell developmental reprogramming, which promote pathogenic colonization. Strikingly, mammalian bacterial pathogens also initiate both effector-triggered susceptibility for cell invasion and effector-triggered immunity via Rho GTPases. In this review, we summarize central concepts of Rho signaling in disease and immunity of plants and briefly compare them to important findings in the mammalian research field. We focus on Rho activation, downstream signaling and cellular reorganization under control of Rho proteins involved in disease progression and pathogen resistance.

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...

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ROPs (rat sarcoma homolog [RHO] of plants) are small monomeric GTPases that function as signalling hubs in cell polarity processes that involve cytoskeleton reorganization (Mucha et al., 2011). Pollen tube growth, the development of epidermal pavement cells and root hairs, but also processes that are important during plant-microbe interactions are examples of ROP-regulated processes (Engelhardt et al., 2020;Zheng & Yang, 2000). ROPs are considered molecular switches due to their ability to shuttle between a signalling-inactive, guanosine diphosphate (GDP)-bound state and a signalling-activated guanosine triphosphate (GTP)-bound state (Bloch & Yalovsky, 2013).An interaction with downstream signalling partners, and therefore signal transduction, only occurs in the GTP-bound state (Nagawa

Key message CRIB motif-containing barley RIC157 is a novel ROP scaffold protein that interacts directly with barley RACB, promotes susceptibility to fungal penetration, and colocalizes with RACB at the haustorial neck. Abstract Successful obligate pathogens benefit from host cellular processes. For the biotrophic ascomycete fungus Blumeria hordei (Bh) it has been shown that barley RACB, a small monomeric G-protein (ROP, Rho of plants), is required for full susceptibility to fungal penetration. The susceptibility function of RACB probably lies in its role in cell polarity, which may be co-opted by the pathogen for invasive ingrowth of its haustorium. However, how RACB supports fungal penetration success and which other host proteins coordinate this process is incompletely understood. RIC (ROP-Interactive and CRIB-(Cdc42/Rac Interactive Binding) motif-containing) proteins are considered scaffold proteins which can interact directly with ROPs via a conserved CRIB motif. Here we describe a previously uncharacterized barley RIC protein, RIC157, which can interact directly with RACB in planta. We show that, in the presence of constitutively activated RACB, RIC157 shows a localization at the cell periphery/plasma membrane, whereas it otherwise localizes to the cytoplasm. RIC157 appears to mutually stabilize the plasma membrane localization of the activated ROP. During fungal infection, RIC157 and RACB colocalize at the penetration site, particularly at the haustorial neck. Additionally, transiently overexpressed RIC157 renders barley epidermal cells more susceptible to fungal penetration. We discuss that RIC157 may promote fungal penetration into barley epidermal cells by operating probably downstream of activated RACB.

Our understanding of obligate biotrophic pathogens is limited by the scarce knowledge concerning the molecular function of virulence factors. We established Arabidopsis thaliana host induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including the CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. In consistence, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic A. thaliana confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function that was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of the widely spread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

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