Barley RIC157, a potential RACB scaffold protein, is involved in susceptibility to powdery mildew.

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. 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.

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f. sp. hordei.

In barley (Hordeum vulgare), signalling rat sarcoma homolog (RHO) of plants guanosine triphosphate hydrolases (ROP GTPases) support the penetration success of Blumeria graminis f. sp. hordei but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of ROP-bound GDP for GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant-specific ROP nucleotide exchanger domain (PRONE). Here, we performed phylogenetic analyses and annotated barley PRONE-GEFs. The leaf epidermal-expressed PRONE-GEF HvGEF14 undergoes a transcriptional down-regulation on inoculation with B. graminis f. sp. hordei and directly interacts with the ROP GTPase and susceptibility factor HvRACB in yeast and in planta. Overexpression of activated HvRACB or of HvGEF14 led to the recruitment of ROP downstream interactor HvRIC171 to the cell periphery. HvGEF14 further supported direct interaction of HvRACB with a HvRACB-GTP-binding CRIB (Cdc42/Rac Interactive Binding motif) domain-containing HvRIC171 truncation. Finally, the overexpression of HvGEF14 caused enhanced susceptibility to fungal entry, while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells during fungal penetration.

An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection.

Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including 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. Consistent with this, 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 Arabidopsis 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, which 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 this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

Regulation and Functions of ROP GTPases in Plant-Microbe Interactions.

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.

Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence.

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.