The molecular dialog between oomycete effectors and their plant and animal hosts

Oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that include some of the most notorious pathogens of plants and animals. Through the deployment of a remarkably diverse array of effector proteins, oomycete pathogens succeed to overcome host defences and cause infection. Effectors can operate extracellularly or enter living cells where they target diverse subcellular compartments. Genome sequence information indicates that oomycetes express several hundred host-translocating effectors potentially targeting a myriad of host processes. To counteract, plants rely on a wide variety of extra-and intracellular immune receptors facilitating pattern-triggered and effector-triggered immunity, respectively. Similarly, effectors from animal pathogenic oomycetes also target host immune response pathways, which in turn causes the activation of the humoral and adaptive immune system. In this review, we compare plant and animal pathogenic oomycete effectors regarding


1.
Oomycetes: ever (re)emerging threats to agriculture, aquaculture and nature Oomycetes evolved approximately 400e800 million years ago from an autotrophic algae-like marine ancestor, and subsequently lost the ability to perform photosynthetic metabolism in adaptation to a heterotrophic lifestyle (Beakes et al., 2012;Matari and Blair, 2014).The lineage of oomycetes, or Oomycota, is constituted by two subclasses: Peronosporomycetidae, comprising most phytopathogenic species; and Saprolegniomycetidae, referred to as "water moulds", including animal pathogenic species (Fig. 1).Many oomycetes are thought to have evolved to a parasitic lifestyle by developing adaptations in their motile stage (Blanco and Judelson, 2005;Cerenius and S€ oderh€ all, 1985) turning them into devastating pathogens of both plants as well as farmed and wildlife populations of aquatic animals.But there are also saprotrophs and opportunistic pathogens of insects, vertebrates, and microbes (Judelson, 2012;Sarowar et al., 2014).
Plant pathogenic oomycetes have an enormous environmental and social impact.In 1845, one million people died and another million emigrated during the Irish potato famine due to the loss of potato crops to the late blight causing agent, Phytophthora infestans.During the late 1970s approximately 10% of total soybean production in Ohio, USA, were lost to Phytophthora sojae only.Other economically important plant pathogenic oomycetes are Plasmopara viticola and Phytophthora citrophthora decimating grapevine and citrus cultures, respectively (Erwin and Ribeiro, 1996), Phytophthora palmivora causing cocoa black pod (Drenth and Guest, 2013), Phytophthora nicotianae with a recognised host range of more than 255 plants worldwide (Panabi et al., 2016), Albugo candida causing white blister rust of Brassica spp.(Choi et al., 2011), and the downy mildew Pseudoperonospora cubensis infecting cucurbits crops worldwide (Savory et al., 2011).Similarly, most animal pathogenic oomycetes have a major impact on freshwater ecosystems, such as Saprolegnia parasitica that infects eggs as well as adult salmonids (van West, 2006).About 10%, but occasionally as high as 50%, of farmed Atlantic salmon suffer from saprolegniosis.Other important oomycetes for aquatic systems are Saprolegnia diclina infecting fish eggs but also insects (van den Berg et al., 2013), Saprolegnia ferax decimating amphibians (Romansic et al., 2009), Aphanomyces astaci causing crayfish plague and Halioticida noduliformans infecting shellfish (Alderman and Polglase, 1986;Muraosa et al., 2009).The list of Phytophthora species and Saprolegniales affecting agriculture and aquatic life, respectively, is extensive and each year new species are discovered worldwide.Economic losses caused by many pathogenic oomycete species are huge due to their broad host range and high adaptability to biotic as well as abiotic stress.
Fig. 1 e Schematic representation of Oomycota subclasses and its orders.Oomycota species can be found in a variety of ecosystems, and have different lifestyles (saprophytic, pathogenic, or obligate).There are about 600 species in 90 genera, the vast majority being plant pathogenic oomycetes within the orders Rhipidiales, Albuginales, Peronosporales and Pythiales with a few exceptions, while animal pathogenic oomycetes are found mostly in the Saprolegniales order.Based on their pathogenic lifestyle, oomycetes are divided into (obligate) biotrophs that depend on a living host to thrive, necrotrophs that kill their host upon infection and feed saprophytically off decaying tissue, or hemibiotrophs that have an initial biotrophic phase followed by a necrotrophic phase (Fawke et al., 2015) (see Rodenburg et al., 2020 for further details).In order to initiate the infection process, pathogens adhere to the host mediated by specialised infection structures or mucilaginous and adhesive substances of spores (Ali and Bakkeren, 2011).Phytopathogenic oomycetes penetrate their host by a combination of degradation of extracellular host defence structures and mechanical force using a specialised structure called appressorium (Bronkhorst et al., 2021;Kebdani et al., 2010).Subsequently, single host cells are penetrated by a haustorium, a specialised hyphal structure that invaginates the host plasma membrane while keeping the plant cell intact.Haustoria play essential roles in suppression of host defences by releasing effector proteins with immunomodulatory functions (Wang et al., 2017;Whisson et al., 2007;Derevnina et al., 2021;Liu et al., 2022) and potentially in nutrient acquisition (Kagda et al., 2020).Appressoria-like structures have also been observed in several fish pathogenic Saprolegnia species upon contact with solid objects such as insect legs or fish cells (Willoughby LG, 1987).However, the infection process in animal pathogenic oomycetes is far less understood compared to plant pathogenic oomycetes.
In order to facilitate the infection process, by overcoming host defences and adapting their metabolism, pathogenic oomycetes secrete effector proteins (Rodenburg et al., 2020).Effector proteins are divided into apoplastic (extracellular) and cytoplasmic (intracellular) effectors.Once secreted, apoplastic effectors act in the extracellular space surrounding host cells, while cytoplasmic effectors translocate inside the host cell (Fawke et al., 2015).Effector proteins generally comprise an N-terminal signal peptide which directs them to the endoplasmic reticulum from where they are secreted.Oomycete apoplastic effectors include numerous hydrolytic enzymes involved in host cell component degradation and various small cysteine-rich proteins that exhibit diverse activities.In contrast, cytoplasmic effectors are translocated into the host cell where they manipulate host processes, suppress host responses or provide nutrients (Bozkurt et al., 2012).While apoplastic effectors are secreted by conventional secretion, the release of cytoplasmic effectors is different (Giraldo et al., 2013;Wang et al., 2017).

2.
Oomycete effector repertoires: a mining experience Genome mining contributes to a better understanding of host pathogen interactions and to date the NCBI database contains 88 oomycete genome sequence assemblies, the vast majority being plant pathogens.The collection includes 61 genomes from Peronosporales, 14 from Pythiales, nine from Saprolegniales, two from Albuginales and two from Lagenidiales (Table S1).Recently, McGowan and Fitzpatrick (2017) analysed 37 complete oomycete genomes for the presence of effector proteins.The computational prediction of effector genes was pioneered by searching for extracellular effectors comprising N-terminal signal peptides in EST databases of P. infestans (Torto et al., 2003).Mining for intracellular effectors became popular immediately after the discovery of the RxLR motif resulting in the identification of the biggest group of cytoplasmic effectors called RxLR effectors (Rehmany et al., 2005;Tyler et al., 2006, Oh et al., 2009).Nevertheless, the identification of effector proteins in animal pathogenic effectors is still problematic due to the lack of conserved motifs or domains (Gaulin et al., 2018;Jiang et al., 2013).However, recently Effec-torO, an algorithm based on lineage specificity, revealed effectors and effector families that were previously missed by only searching for specific features (Nur et al., 2021).Machine learning models such as EffectorP 3.0 (Sperschneider and Dodds, 2022) are trained to predict apoplastic and cytoplasmic effectors from fungi and oomycetes, representing the most recent effector prediction tool to date.
Below, we discuss some representatives of apoplastic and cytoplasmic effectors for both plant and animal pathogenic oomycetes.

2.1.
Apoplastic effectors: the first line of attack In plants, the apoplastic space between the pathogen and the host is acidic and highly enriched in proteases and receptors for defence.Hence, as a counter defence, pathogens have evolved effectors that compromise such host defence mechanisms like small cysteine-rich (SCR) proteins including elicitins, PcFs (first introduced as phytotoxins), and Necrosisand ethylene-inducing protein 1-like proteins (NLP), enzyme inhibitors as well as extracellular proteases for degradation of host structures.SCRs.SCR proteins are relatively small with a high proportion of highly conserved cysteines that form intramolecular bridges resulting in a highly stable, globular protein providing protection against the harsh environment of the extracellular space.However, other canonical protein domains or motifs are missing in this effector class.So far, the exact biological function and/or target of many SCR proteins is unknown despite evidence of their importance in facilitating the infection process (Zhang et al., 2021;Chen et al., 2016;Orsomando et al., 2011).
Elicitins are conserved SCR proteins exclusive to oomycetes and occur as complex multigene families in plant pathogenic Phytophthora and Pythium species (Derevnina et al., 2016;Jiang et al., 2006) and animal pathogenic oomycetes such as Py.insidiosum or Aphanomyces (McGowan and Fitzpatrick, 2017).They are important for the survival of sterol auxotroph phytopathogens by extracting sterols and membrane lipids from the plant membrane as an external lipid source (Dahlin et al., 2017;Derevnina et al., 2016).Similarly, ELI025 from the animal pathogenic Py. insidiosum has been predicted to act as a sterol-carrying protein which could compensate for the sterol auxotroph lifestyle of Pythium (Lerksuthirat et al., 2015).Sterols and fatty acids also facilitate sexual reproduction and oospore production in Phytophthora and therefore, elicitins can indirectly contribute to genetic variation which potentially increases the virulence of certain strains due to higher selective pressure resistance (Chepsergon et al., 2020).The incomplete sterol biosynthesis pathway in Py. insidiosum is conferring antifungal drug insensitivity (Lerksuthirat et al., 2017).The biological importance of elicitins for the pathogen as well as high structural conversation and expression during host pathogen interaction show that elicitins share features of microbe-associated molecular patterns (MAMPs) which can be recognised by receptors and induce an immune response in plants (Derevnina et al., 2016;Du et al., 2015).
PcF represents another family of SCR proteins that trigger immune responses in various plant species, such as tomato and strawberry (Nicastro et al., 2009;Orsomando et al., 2003).PcF was first described as a phytotoxin in P. cactorum isolated from infected Fragaria and bioinformatic analysis showed that this effector family is exclusive to Peronosporales (Orsomando et al., 2001;Lin et al., 2020) (Fig. 2).One subclass, called SCR74, is under strong positive selection and its coevolution with the host has expanded this gene drastically in P. infestans resulting in up to 17 variants (Lin et al., 2020).Also, knock out studies of PcF homologues SCR82 or SCR96 of P. capsici and P. cactorum, respectively, resulted in reduced virulence on solanaceous hosts (Chen et al., 2016;Zhang et al., 2021).The high expression during infection, gene expansion, as well as the induction of a plant immune response, indicates an important role of SCR74 proteins for the hostpathogen interaction (Lin et al., 2020;Liu et al., 2005).
in hemibiotrophic oomycetes and fungi revealed that only NLPs expressed during infection show cytotoxicity indicating an additional role of NLPs at other life stages (Dong et al., 2012;Santhanam et al., 2012).

Apoplastic enzymes
Oomycetes have accumulated a large number of hydrolytic enzymes (Fig. 2).In the apoplast, hydrolytic enzymes are important contributors to host invasion through degradation of host macromolecules such as sugars, proteins, lipids or cutin/chitin as well as pathogenesis by altering host physiology (Haas et al., 2009;Tyler et al., 2006).
Glycoside hydrolases (GHs, also glucosidases) belong to the group of carbohydrate-active enzymes (CAZymes) that catalyse the hydrolysis of glycosidic bonds in complex sugars.In a first line of attack to invade the host and establish an infection, GH degrade sugar moieties at the host surface, hence these enzymes are abundantly secreted by all oomycetes and the repertoire is likely linked to the oomycete lifestyle.GHs are most commonly found in Phytophthora (Fig. 2) (Ospina- Giraldo et al., 2010;McGowan et al., 2020).Some families of GHs are abundant across all oomycetes (e.g.GH3), whereas other families are unique to certain taxonomic groups, e.g.GH12 in Phytophthora (McGowan and Fitzpatrick, 2017).In contrast to Phytophthora, Pythium and Hyaloperonospora seem to have a significantly reduced repertoire of GHs (Zerillo et al., 2013;Brouwer et al., 2014).Beside cellulose and pectin, hemicellulose is an important part of the primary cell wall of all land plants and hence, phytopathogenic oomycetes secrete xyloglucan-specific endoglucanases to promote host structure degradation (XEG1, GH12).The soybean pathogen P. sojae, secretes PsXEG1 at early stages of infection when it acts as an important virulence factor (Ma et al., 2015;Yoshizawa et al., 2012).However, PsXEG1 is degraded by the host aspartic protease GmAP5, which is reduced after N-glycosylation of PsXEG1.To further increase XEG1 activity in the apoplast, a PsXEG1 paralogue (PsXLP1) which is protected against GmAP5 proteolysis by a C-terminal deletion is secreted to reduce total GmAP5 activity (Xia et al., 2020) (Fig. 3A).This continuous arms race for physiological dominance is driving the (co-) evolution of host defence and virulence genes in the apoplast.However, plant pathogenic oomycetes do not just secrete cell wall hydrolysing enzymes that act as virulence factors such as xyloglucanases (Ma et al., 2015), cellulases (Blackman et al., 2015), glucanases (Anasontzis et al., 2019) and pectinases (Fu et al., 2015;Yang et al., 2018) but also cell wall modifying enzymes such as Pectin acetylesterases (PAEs).They catalyse the deacetylation of pectin, a major cell wall component in higher plants (Kong et al., 2019).PAEs genes are mainly found in Pythiales and Peronosporales (excluding H. arabidopsidis), with a single protein or total absence in Albuginales and Saprolegniales (McGowan and Fitzpatrick, 2017) (Fig. 2).Interestingly, pectin acetylation is important for signalling during biotic stress whereas pectin hypoacetylation usually confers resistance against pathogens (Manabe et al., 2011;Pogorelko et al., 2013;Randoux et al., 2010).Hence, the exact role of pectin acetylesterases secreted by oomycetes during infection requires further investigation.Beside glycolytic hydrolases, plant as well as animal pathogenic oomycetes possess a large repertoire of secreted proteases that cleave peptide bonds.Several protease families have been found in oomycete secretomes including serine proteases (subtilases), cysteine-and aspartic proteases as well as metalloproteases, and reported to be involved in establishing infections (Davis et al., 2006;Jiang et al., 2013;Kiselev et al., 2022;Majeed et al., 2017;Meijer et al., 2014;Schoina et al., 2021).Members of the order Saprolegniales contain most proteases with at least over 150 enzymes for each species (McGowan and Fitzpatrick, 2017) (Fig. 2).S. parasitica secretes SpSSP1, a subtilisin-like serine protease, that is recognised as an antigen by rainbow trout (Oncorhynchus mykiss) serum (Minor et al., 2014).SpSSP1 has potentially an important role in the suppression of host immune responses as it can degrade trout immunoglobulins (Jiang et al., 2013).Also, other oomycetes express SpSSP1 homologues (A.astaci, L. giganteum, P. infestans, P. coralinianum) but their role during host-pathogen interaction is unclear so far.A serine protease secreted by A. invadans has also been shown to be important for the infection of dwarf gourami fish since its mutation results in reduced pathogenicity of A. invadans (Majeed et al., 2017(Majeed et al., , 2018)).The plant pathogenic P. parasitica secretes the cysteine proteases PpCys44/45 and PpCys69 that trigger NPKI-dependent cell death in Nicotiana species and promote pathogen virulence (Zhang et al., 2020).Cysteine proteases were also reported in the secretome of A. invadans (Iberahim et al., 2020;Majeed et al., 2017) or P. infestans (Meijer et al., 2014) which are also up regulated during infection of tomato (Zuluaga et al., 2016).Similar to cysteine proteases, some metalloproteases also show infection-related expression in plant as well as animal pathogenic oomycetes and thereby potentially contribute to pathogen virulence (Schoina et al., 2021).Despite the enrichment of aspartic proteases in Phytophthora (Kay et al., 2011), and the active secretion of at least one of them (Meijer et al., 2014) their potential as virulence factors is yet to be investigated.Although, being omnipresent in all pathogenic oomycetes, proteases and their exact role during infection are less well understood compared to other apoplastic effectors.
Phospholipases are enzymes that hydrolyse phospholipids into fatty acids and other lipophilic molecules.Besides their essential role in intracellular signal transduction through free Ca 2þ and lipid mediators, Phospholipase D (PLDs)-like genes are also described as virulence determinants in P. infestans and P. capsici (Meijer et al., 2019;Nespoulous et al., 1999).PLD-like genes are expressed in germinating cyst as well as during infection eliciting cell death.However, it is unclear if the contribution to virulence occurs from intracellular signalling events or lipid re-arrangement in the host cell.
Oomycetes secrete host-specific enzymes such as cutinases or chitinases, that hydrolyse cutin, a major component of plant cuticles, and chitin, found in insects, invertebrates and fish.In line with the hypothesis of host-pathogen adaption, cutinases are absent in fish pathogenic Saprolegniales but enriched in Phytophthora during infection of plants (Brouwer et al., 2014;McGowan and Fitzpatrick, 2017).In contrast, chitinases are found in Saprolegniales and Pythiales; the crayfish plague pathogen A. astaci possesses the highest total number of chitinase genes (McGowan and Fitzpatrick, 2017;Sabbadin et al., 2021;Shen et al., 2020) (Fig. 2).
In summary, oomycetes exploit a vast repertoire of enzymes in the apoplast to contribute to virulence in plant as well as animal hosts (Figs. 2 and 3).So far, the knowledge Oomycete effectors and host immunity  4) Another RXLR effector, PsAVH18/PSR1, from P. sojae translocates into the plant nucleus and may interact with DCL1 proteins to disrupt biogenesis of miRNAs that target plant defence related genes; as a consequence the NLR-based immunity can be suppressed.( 5) about plant pathogenic oomycetes apoplastic enzymes surpasses the understanding of extracellular effectors in animal pathogenic oomycetes.

Enzyme inhibitors
Similar to oomycetes, also their hosts secrete hydrolases as a first line of attack (Gasteiger et al., 2017;Gong et al., 2019;Wang et al., 2020).In response, oomycetes secrete enzyme inhibitors to overcome those defence mechanisms and thereby drive host-pathogen co-evolution.Plants secrete b-1,3endoglucanases (also pathogenesis-related (PR) protein) to degrade oomycete cell walls to reduce pathogen invasion and/or for the release of glucan elicitors or damageassociated molecular patterns (DAMPs) that subsequently activate plant immunity.For protection, many Phytophthora secrete glucanase inhibitor proteins (GIPs) that specifically inhibit endoglucanase activity in the apoplast (Damasceno et al., 2008;Johnson George et al., 2016;Martins et al., 2014;Rose et al., 2002).For example, soybean produces the apoplastic glucanase inhibitor protein, GmGIP1, which binds to PsXEG1 (xyloglucan-specific endoglucanases from P. sojae) to inhibit its enzymatic activity and thereby facilitates protection.Ultimately, the pathogen counteracts and secretes PsXLP1, a paralogous decoy of PsXEG1 that binds more tightly to GmGIP1 and therefore indirectly increases PsXEG1 activity (Ma et al., 2017) (Fig. 3a).Oomycetes also employ inhibitors of extracellular proteases secreted by the host.Serine proteases inhibitors are characterised by Kazal-like domains and are omnipresent in plant as well as animal pathogenic oomycetes (McGowan and Fitzpatrick, 2017;Tian et al., 2004) (Fig. 2).The serine protease inhibitors EPI1 and EPI10 (two and three Kazal domains, respectively) are secreted during infection by P. infestans to inhibit the pathogenesis-related subtilisin-like serine protease P69B of tomato (Tian et al., 2004(Tian et al., , 2005)).However, proteases are not only secreted to degrade pathogen macromolecules but also to induce plant immunity by specific cleavage of apoplastic effectors.Therefore, serine protease inhibitors such as EPI1 also impair effector-triggered immunity (Wang et al., 2021b).Cysteine protease inhibitors comprise a cystatin-like domain and are less abundant in oomycetes than serine protease inhibitors (McGowan and Fitzpatrick, 2017).P. infestans secretes a whole family of cysteine protease inhibitors, EPIC1-4.EPIC2B interacts and inhibits PIP1 (papainlike cysteine protease Phytophthora Inhibited Protease 1) which is closely related to the tomato apoplastic cysteine protease Rc3 which acts in fungal resistance and is targeted by the protease inhibitor AVR2 of Cladosporium fulvum (Tian et al., 2007).Interestingly, EPIC1 and EPIC2B do not show any sequence homology to AVR2a from C. fulvum but also inhibit Rcr3 (Song et al., 2009).This shows that not only effectors secreted by unrelated pathogens can target the same defence protease but, vice versa, the same inhibitors also target multiple proteases such as EPIC1 and EPIC2B inhibition of the papain-like cysteine protease C14 from potato and tomato (Kaschani et al., 2010).Phytophthora mirabilis secretes the P. infestans EPIC1 orthologue PmEPIC1 and interestingly, due to host adaption each protease inhibitor is more effective against the protease from their respective hosts (Dong et al., 2014).These studies provide another intriguing example of enzyme inhibition as an important counter defence strategy in plant pathogens.

Cytoplasmic effectors: oomycetes in stealth mode
In a second line of attack, oomycetes secrete cytoplasmic effectors that require translocation inside the host to reach their intracellular target.Once inside they can overcome further host defence mechanisms and manipulate host processes for the pathogens benefit.Plant pathogenic oomycetes from the Phytophthora genus and downy mildews secrete two large classes of cytoplasmic effectors, namely crinkling and necrosis (CRN) effectors and RxLR effectors.Both classes of effectors possess an N-terminal signal peptide for secretion, unique motifs for their correct translocation and a C-terminal effector domain important for their function (Amaro et al., 2017;Schornack et al., 2010;Whisson et al., 2007).So far, CRN and RxLR effectors seem to be exclusively enriched in plant pathogenic oomycetes.

CRN effectors: intimate with the host
The CRN protein family is widespread amongst oomycetes and thought to be more ancient than RxLR effectors ( Schornack et al., 2010;Stam et al., 2013c;Zhang et al., 2016).CRN-coding genes are present in the genome of all plant pathogenic oomycetes sequenced to date, including Hyaloperonospora and basal Aphanomyces spp.but not Eurychasma dicksonii (Amaro et al., 2017;Gaulin et al., 2008;Grenville-Briggs et al., 2011), while Albuginales and Saprolegniales encode for only one CRN effector (McGowan and Fitzpatrick, 2017) (Fig. 2).Since CRN effectors are generally highly expressed and some of them up-regulated during infection, they potentially play an important role for the virulence (Haas et al., 2009;Shen et al., 2013).CRN proteins comprise an N-terminal signal peptide, a LXLFLAK motif and a highly conserved "HVLVxxP" that separates the N-terminus from the C-terminus (McGowan and Fitzpatrick, 2017;Stam et al., 2013b).Almost all CRN effectors accumulate in the nucleus when transiently overexpressed in planta which is essential for their function of targeting host nuclear processes (Stam et al., 2013a).CRN13 from Aphanomyces euteiches and BdCRN13 from Batrachochytrium dendrobatidis contain an endonuclease HNH-like motif that allows the protein to interact with host DNA and trigger DNA damage repair response, which promotes host susceptibility (Ai et al., 2021;Ramirez-Garc es et al., 2016).PsCRN108 from P. sojae also contains a DNA-binding HNH-motif and inhibits heat shock protein (Hsp) gene expression at the transcription level in A. thaliana, N. benthamiana and soybean (Song et al., 2016).Similarly, CRN12_997 from P. capsici binds to a transcription factor, SlTCP14-2, in tomato which causes inhibition of immune responses and enhances susceptibility of the host plant (Stam et al., 2021).The insect pathogen Pythium guiyangense also possesses CRN effectors which are toxic to insect cells (Shen et al., 2019).Interestingly, Py. guiyangense CRN proteins showed sequence divergence of at least 50% with the closest CRN protein from any plant pathogenic Pythium species indicating that CRN proteins are highly divergent between insect and plant pathogenic Pythium species, and probably adapting to different host nuclear processes.
However, CRN effectors do not only regulate DNAdependent processes in the nucleus.PsCRN78 from P. sojae does not localise to nuclei and interacts with PIP2-family aquaporin proteins including NbPIP2; 2 from N. benthamiana and GmPIP2-13 from soybean which are located at the plant plasma membrane (Ai et al., 2021).The P. infestans effector PiCRN8 shares high sequence similarity with serine/threonine kinases and suppresses plant defence as well as causes cell death (Van Damme et al., 2012).Also, the P. sojae-encoded CRN proteins PsCRN63 and PsCRN115 manipulate host hydrogen peroxide homeostasis and promote pathogenicity by direct interaction with plant catalases (Zhang et al., 2014).
Taken together, the high divergence and functional dispersion of CRN effectors enables them to manipulate various plant defence mechanisms.Nevertheless, they remain relatively understudied and more is still to be discovered (Chen et al., 2018;Maximo et al., 2019;Xiang et al., 2021).

RXLR effectors: Pas de deux
The most studied group of cytoplasmic effectors of plant pathogenic oomycetes are the RxLR effectors.They are characterised by an N-terminal signal peptide, followed by an Arg-X-Leu-Arg (RxLR) motif often linked with an Asp-Asp-Arg (EER) motif (Bhattacharjee et al., 2006;Rehmany et al., 2005;Whisson et al., 2007).Variants of the RxLR motif are present in effectors of downy mildews such as GKLR in B. lactucae (Stassen et al., 2013) or RxLK in P. halstedii (Sharma et al., 2015).Downstream of the RxLR/EER motif, RxLR effectors usually possess a WY domain.The WY domain contains conserved W, Y, and L motifs (Jiang and Tyler, 2012) forming an alpha-helical fold which might be important for regulating effectoretarget interactions.The WY domain is specific to the Peronosporales and was predicted to be present in nearly half of the RxLR effectors from P. infestans and 25 % of the RxLR effectors in H. arabidopsidis (Wood et al., 2020), but is considerably less represented in other oomycete orders.However, effectors from downy mildew such as P. viticola or B. lactucae comprise a conserved WY domain but lack the N-terminal RxLR or other short motifs (Combier et al., 2019;Wood et al., 2020).Despite the progress in the detailed understanding of the function of cytoplasmic effectors from plant pathogenic oomycetes, their delivery into host cells has rarely been directly visualised and is poorly understood (Wang et al., 2017).The mechanisms by which RxLR effectors enter the host cell has been under debate for a decade (Kale and Tyler, 2011;Wawra et al., 2012) and were proposed to involve phosphoinositol-3-phosphate (PI3Ps) binding (Kale and Tyler, 2011), tyrosine-o-sulphate dependent translocation (Wawra et al., 2012) or even translocon proteins in combination with chaperones (Bozkurt et al., 2012).However, recently it was shown that the RxLR motif of AVR3a from P. infestans is cleaved before secretion from the pathogen and thereby is likely not involved in host translocation (Wawra et al., 2017).
In contrast to CRN effectors, RXLR effectors target various subcellular compartments of host cells to perform their functions including the plasma membrane, endoplasmic reticulum, mitochondria, cytoplasm, or nucleus (Boevink et al., 2016;Wang et al., 2018a).So far, 30 RxLR effectors from plant-pathogenic oomycetes have been studied and their target identified (He et al., 2020).About 50% of RxLR effectors with known targets affect transcription and signalling pathways which are often important for the regulation of the immune response, but also host proteins involved in protein stability, RNA processing, general metabolism and cellular trafficking (Petre et al., 2021;Wang et al., 2021a) are affected by RxLR effectors during an infection.
RxLR effectors have developed various mechanisms to affect their intracellular target.Due to their limited size, RxLR effectors rarely comprise domains with enzymatic activities.Therefore, only PsAVR3b from P. sojae has been shown to comprise a hydrolase domain so far, activated by a host peptidyl-prolyl isomerase, that negatively regulates plant immunity in soybean (Dong et al., 2011;Kong et al., 2015).However, more common amongst effectors is the modulation of their targets' activity.This includes the inhibition of positive regulators of immunity such as mitogen-activated protein (MAP) kinases by PexRD2 and Pi22926 from P. infestans (King et al., 2014;Ren et al., 2019) or the peptidyl-prolyl isomerase (PPIase) activity by PcAVR3a12 from P. capsici (Fan et al., 2018).Also, the negative regulators of immunity are modulated by RxLRs, such as the protein phosphatase (PP1c) by Pi04314 (Boevink et al., 2016).The central immune kinase RLCK-VII (receptorlike cytoplasmic kinase subfamily VII) is targeted by PcRxLR25 from P. capsici and its phosphorylation pattern changes upon binding which affects RLCK-VII activity (Liang et al., 2021).Another mode of action is affecting the stability of host proteins by protein degradation as for the transcription factor MED19a after interaction with HaRxL44 from H. arabidopsis (Caillaud et al., 2013) or the 1-aminocyclopropane-1carboxylic acid synthase by the P. sojae effector PsAVH238 (Yang et al., 2019); or by protein stabilisation as for binding immunoglobulin proteins (BiPs) by PsAVH262, for S/K/R-rich proteins (SKRPs) bound by PsAVR3c, or for the E3 ligase GmPUB13 stabilized by AVR1d, all secreted by P. sojae (Huang et al., 2017;Jing et al., 2016).In addition, RxLR effectors also disrupt the formation of host protein complexes and thereby affect downstream processes.The P. capsici effector PcAVH103 disrupts EDS1-PAD4 complex formation thereby suppressing immune signalling pathways (Li et al., 2020) and PexRD54 from P. infestans hijacks autophagosomes by abolishing the interaction between ATG8CL and Joka2 (Dagdas et al., 2016(Dagdas et al., , 2018)).While protein degradation is also a form of relocalisation, some effectors also prevent targets from reaching their natural localisation.This has been observed for PcRxLR48 from P. capsici, facilitating the nuclear accumulation of NPR1 (Li et al., 2019a), PsAVH52 from P. sojae directing the transacetylase TAP from the cytoplasm to the nucleus (Li et al., 2018) and Pi03192 from P. infestans preventing the nuclear localisation of NAC transcription factors (McLellan et al., 2013).
Another effector protein from S. parasitica, host targeting protein 3 (SpHTP3), in addition to a signal peptide for secretion and an effector domain with nuclease activity, also comprises an RxLR motif (Trusch et al., 2018).In contrast to SpHTP1, selftranslocation of SpHTP3 is RxLR-independent and mediated by a C-terminal basic helix.The C-terminus of SpHTP3 interacts with the negatively charged cell of the host before complex formation with gp96 in lipid-rafts.A similar host membrane interaction was observed for negative patches on the surfaces of AVR3a from P. infestans and AVR1b from P. sojae but their function remains to be revealed (Trusch et al., 2018;Wawra et al., 2012;Yaeno et al., 2011).Interestingly, the SpHTP3 homologue PsHTP3 from the plant pathogenic P. sojae also translocates in a pathogen-independent manner into non-host cells (Fig. 3B).
To date, research into RxLR effector proteins is more focused on those that target host defence mechanisms in one way or another (Anderson et al., 2015;He et al., 2020;Naveed et al., 2020).Hence, many studied RxLR effectors are reported to suppress PAMP-triggered (PTI) and effector triggered immunity (ETI) which is caused by ligand detection by immune receptors of the plant.To the best of our knowledge, all known characterised Avr genes from oomycetes belong to the family of RxLR effectors and typically exhibit elevated expression at early stages of infection, occur in gene-parse regions of the genome and are subject to accelerated evolution (Haas et al., 2009;Vleeshouwers et al., 2011).In addition to their involvement in immunity, RxLR effectors target many cellular processes that are important during a host-pathogen interaction.

3.
Hosts: what to do when under attack While distinct oomycetes exploit similar mechanisms to infect their hosts, the defence mechanisms deployed by animal versus plant hosts is remarkably different.Animals use innate as well as adaptive immune responses, while plants lack a circulating immunity and rely solely in an innate immune system built on complex network of signalling pathways to defend themselves against pathogenic oomycetes (Fig. 4).

Plant immunity
In plants, the cell wall is the first important physical barrier against any invading pathogens; often reinforced by lignification, suberization and callose (b-1,3-glucan) deposition to prevent or at least slow down the infection process.Callose deposition is often found during early stages of infection, mainly in incompatible hosteoomycete interactions verifying the efficiency of such a process (Bouwmeester et al., 2011;Fabro et al., 2011;Huitema et al., 2003;Wang et al., 2013).
In addition to physical barriers, plants have a phytohormone signalling network as a universal defence response.Phytohormones such as jasmonic acid (JA), salicylic acid (SA) and ethylene, are key factors of plant immune responses.SA is a central regulator required for the hypersensitive response in plants (Halim et al., 2006) (Fig. 4).
A highly specific layer of protection is the receptor-based plant immune system that is divided in two lines of defence.One is a general system called pattern-triggered immunity (PTI) based on pattern recognition receptors (PRRs) in the plasma membrane that recognise MAMPs, DAMPs or apoplastic effectors (Dodds and Rathjen, 2010;Jones and Dangl, 2006).The second system is called effector-triggered immunity (ETI) based on intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognise effectors and neutralise their effects.ETI responses are stronger than PTI and often result in a localised hypersensitive response (HR).HR is an induced, very local cell death to avoid spread of pathogens that require living tissue for successful colonisation (Cesari, 2018;Cui et al., 2015;Fei et al., 2016) (Fig. 4).

PTI: PRR-based apoplastic immunity in plants
PRRs (pattern recognition receptors) are divided into two classes, based on the presence or absence of a cytosolic kinase domain.The receptor-like kinases (RLKs) contain an extracellular domain, a transmembrane domain and the kinase domain.The receptor-like proteins (RLPs) have the same domain architecture excluding the intracellular kinase domain, and therefore are often associate with RLKs to transduce ligand perception into intracellular signalling (Monaghan and Zipfel, 2012).This was shown for LRR-RLPs associating with SUPPRESSOR OF BIR1-1 (SOBIR1) or SOBIR1like LRR receptor kinase to form a bimolecular equivalent of a genuine RLK (Gust and Felix, 2014).Additionally, the BRASSI-NOSTEROID INSENSITIVE 1-associated receptor kinase (BAK1) is a key positive regulator of immunity that is recruited to RLPs and RLKs upon ligand perception (Zipfel, 2014).So far, only few plant PRRs that recognize oomycetes MAMPs or apoplastic effectors have been identified.
The first RLP specifically recognising elicitins of Phytophthora, is the elicitin receptor (ELR) encoded by the wild potato Solanum microdontum.Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans (Du et al., 2015).ELR associates with BAK1/SERK3 and with SOBIR1 functioning as an adaptor kinase (Domazakis et al., 2018).Another LRR-RLP, RLP23 from Arabidopsis thaliana, induces an immune response after binding to a conserved motif in necrosis and ethylene-inducing peptide 1-like proteins (nlp20) in vivo.Similarly to ELR, RLP23 forms a complex with SOBIR1 and recruits BAK1 after ligand binding (Albert et al., 2015).The transfer of RLP23 to potato (Solanum tuberosum) confers nlp20 recognition and enhanced resistance to P. infestans (Albert et al., 2015).Later, using a high-throughput approach, the LRR receptorlike protein response to XEG1 (RXEG1) was identified in Nicotiana benthamiana (Wang et al., 2018b) (Fig. 3A).RXEG1 associates with XEG1 via the LRR domain and forms a complex with BAK1 and SOBIR1.Overexpression of RXEG1 in N. benthamiana significantly reduces lesions caused by P. parasitica, thereby contributing to plant immunity (Wang et al., 2018b).Other well-known MAMPs and apoplastic effectors, such as Pep13 (peptide fragment of a cell wall glycoprotein GP42), CBEL (cellulose binding elicitor lectin), PcF and SCR74, still require identification of their corresponding PRRs and recognition mechanism.

ETI: NLR-based cytoplasmic immunity in plants
Genetic resistance to oomycetes in plants was discovered based on the hypersensitive response (HR) (Kamoun et al., 1999).In the past two decades, it became clear that ETI based on resistance (R) genes of the NLR class is a powerful HRdefence against oomycetes (Jones and Dangl, 2006).NLR plant receptors are multidomain proteins that consist of a central nucleotide-binding (NB-ARC) domain followed by a leucine-repeat (LRR) domain and an N-terminal domain that determines their specific signalling role.These N-terminal domains either belong to the group of Toll-interleukin 1 receptor (TIR) domains, coiled-coil (CC) domains or RNL domains (Cui et al., 2015;Jones et al., 2016;Tamborski and Krasileva, 2020).
To identify and clone resistance genes, an effectoromics approach was pioneered in potato by exploiting effectors from P. infestans in high throughput functional screens in wild Solanum germplasm (Vleeshouwers et al., 2011).In this routine, several NLR genes were identified from a variation of wild potato species.Most of them belong to the CC-NLR family and recognise avirulence (AVR) proteins from P. infestans.For example, R3a and Rpi-blb2 from Solanum demissum and Solanum bulbocastanum recognise the RXLR effectors AVR3a and AVRblb2, respectively (Huang et al., 2004;Oh et al., 2014;van der Vossen et al., 2005).Similarly, CC-NLR resistance genes described in soybean play the same role but against P. sojae indicating host specificity (Bhattacharyya et al., 2005;Dong et al., 2011;Shan et al., 2004).Beyond the R-Avr matching pairs, effector recognition, either directly or indirectly, is mediated via the LRR domain (Caplan et al., 2008;Rairdan and Moffett, 2006).A recent study described how two allelic variants from the S. chacoense resistance gene Rpi-chc1 recognise different RXLR effectors from the PexRD12/30 family in P. infestans through their LRR domain (Monino-Lopez et al., 2021;Vossen et al., 2011).Plant NLR receptors are widespread because of the continued (co-) evolutionary pressure caused by secreted Avr genes from the pathogen.Due to the evolutionary transition that some NLR receptors have experienced, they can play different roles when it comes to effector recognition.Therefore, they are more likely to function in pairs or multimers (Stassen & Van den Ackerveken, 2011;Tamborski and Krasileva, 2020;Tyler, 2008).
The NLR receptors are activated by the NB-ARC domain after the binding of ATP followed by oligomerisation.Receptor oligomerisation is essential for their activation and was first described on structural level in the CC-type NLR ZAR1 (HOPZ-activated resistance 1) receptor (Wang et al., 2019) and the TIR-NLR Roq1 (recognition of XopQ1) receptor (Martin et al., 2020).The ZAR1 resistosome forms a pentamer via the a-helix and is activated through the a 1 -helix in the N-terminus (Wang et al., 2019).In contrast, Roq1 forms a tetramer complex via its TIR domains and is activated by the BB-loop with NADase activity (Martin et al., 2020).
The two-tiered immune system of cell-surface and intracellular immunity has recently been revised to a model of mutual potentiation of PTI and ETI, and conceptionally unites the two layers to synergistically activate defence (Ngou et al., 2021;Yuan et al., 2021).Moreover, receptor networks are shown to be highly interconnected to phytohormone signalling pathways, and all together enable activation of strong defences against plant pathogens (Ngou et al., 2022).

Animal response
The host response against animal pathogenic oomycetes is best studied in teleosts (bony fish), in particular salmonids (Elameen et al., 2021;Hussein et al., 2001;van West, 2006).Fish possess an innate as well as an adaptive immune response like other vertebrates (Tort et al., 2003).The innate immune system includes primary defence barriers, such as mucus and epidermis, and cellular processes, such as phagocytosis and production of antimicrobial lytic factors as a humoral component (Bayne and Gerwick, 2001).The importance of a physical barrier as an efficient defence mechanism was demonstrated by the chorion of ova of Atlantic salmon (Salmon salar) which gives better protection against S. parasitica when thicker (Songe et al., 2016).Cell and humoral responses act complementary in synchrony for the efficient recognition of potential pathogens (Canesi and Proch azkov a, 2014).For example, free circulating blood cells such as haemocytes or coelomocytes produce ROS in response to diverse microorganisms (Becking et al., 2015;Canesi and Proch azkov a, 2014) (Fig. 4) as seen by the resistant crayfish (Pacifastacus leniusculus) in response to Aphanomyces astaci (Becking et al., 2015).Also, the phenoloxidase system is an important innate immune mechanism in invertebrates, consisting of a cascade of proteins and serine proteases recognising diverse microorganism molecules resulting in melanisation of the pathogen but also tissue damage (Gonz alez-Santoyo and C ordoba-Aguilar, 2012; Lu et al., 2014).A rapid increase of phenoloxidase activity in the haemolymph was observed in resistant crayfish (P.leniusculus) compared to the susceptible noble crayfish (Astacus astacus) after infection with A. astaci resulting in the encapsulation of infection structures into a sheath of melanin and thereby preventing pathogen growth (Becking et al., 2015;Cerenius et al., 2003).Altogether, the innate immune system plays a crucial role in fighting oomycetes infections, in particular fish infection with A. invadans (Kumaresan et al., 2018;Yadav et al., 2014Yadav et al., , 2016)).
While the immune system of invertebrate species is based exclusively on innate immunity to counteract invading pathogens (Canesi and Proch azkov a, 2014;Kvell et al., 2007), vertebrates comprise an additional, more advanced adaptive immune system that is activated in response to a particular pathogen once the first line of defence is overcome (Kiron, 2012).Hence, the adaptive immune response is highly complex, specific and characterised by diversity and memory (Rauta et al., 2012).It plays a crucial role in protection against reinfections by creating memory cells and specific soluble and membrane-bound receptors such as T-cell receptors and immunoglobulins (Ig, antibodies) allowing a fast and efficient response to a reoccurring pathogen.
Experiments with fish cell lines challenged with Achlya bisexualis or S. parasitica in vitro, indicate that cytokines (TNFa, IL-8, IL-1b, IL-11) as well as parts of the antigen presenting system (TAP, major histocompatibility complex (MHC-I) chaperone) involved in inflammatory response are upregulated (de Bruijn et al., 2012;Kales et al., 2007;Roberge et al., 2007).Simultaneously, antimicrobial peptides (hepcidin and cathelicidin) and other components of the innate immune response (COX-2, CD209a and b) are more highly expressed during an infection.A strong inflammatory response in vitro is not only seen in the presence of the whole pathogen but also with cell wall components initiating a Th-2 like response (Belmonte et al., 2014).Similarly, the glucan extract from Pythium insidiosum induces a specific Th1/Th17 cellular immune response in BALB/c mice (Ledur et al., 2018;Tondolo et al., 2017Tondolo et al., , 2020)), and cytokines up regulated in human corneal epithelial cells and monocyte derived macrophages infected with Py. insidiosum (Wongprompitak et al., 2018).In vivo studies have shown that brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) are able to produce neutralising antibodies against S. parasitica (Fregeneda- Grandes et al., 2007;Minor et al., 2014).Interestingly, fish infected with S. parasitica show a low antibody titre, suggesting a possible suppression of the antibody response by S. parasitica (Fregeneda-Grandes et al., 2009).However, it also has been shown that the purinergic signalling pathway in the spleen of grass carp (Ctenopharyngodon idella) enters a selfsustained pro-inflammatory deleterious cycle, contributing to an enhanced inflammatory process during saprolegniosis (de Freitas Souza et al., 2019).Similarly, analysis of the head kidney of common carp (Cyprinus carpio) experimentally infected with A. invadans revealed an immune response comprising differentiated gene expression in 21 immune pathways, the activation of the NLRP3 inflammasome, enhanced phagocytosis and increased recruitment of leukocytes to the site of infection (Verma et al., 2020).
Interestingly, the nematode Caenorhabditis elegans induces chitinase-like genes in its epidermis resulting in the modification of its cuticle that reduces the attachment of the oomycete Myzocytiopsis humicola and thereby delays the infection process (Grover and Barkoulas, 2021;Osman et al., 2018).This mechanism resembles the callose deposition in the plant cell wall (Fabro et al., 2011).
Understanding the host immune response to oomycetes also comprises potential for new disease control strategies.For example, LBP/BP1 (a lipopolysaccharide binding protein/ bactericidal permeability increasing protein) highly expressed in eggs of the freshwater snail Biomphalaria glabrata, shows biocidal activity against S. parasitica as well as S. diclina (Baron et al., 2013).

Conclusion/Final remarks
As a taxon of successful pathogens that colonise diverse host taxa, oomycetes have evolved many virulence traits to adapt to new hosts, colonize different tissue types and cope with a diversity of host defence mechanisms.In comparison to animal pathogenic oomycetes, the genomes of plant pathogenic Phytophthora species encode larger numbers and higher variation of RXLR and CRN effectors, hydrolases and enzyme inhibitors.This reflects the importance of these gene families to suppress MAMP-triggered immunity, manipulate the plant immune response and counterattack plant proteases.In contrast, animal pathogenic oomycete genomes suggest adaptations to an animal pathogenic lifestyle by the loss of plant cell wall degrading enzymes and NLP proteins as well as expansions of chitinases, cysteine proteases and subtilases.Animal pathogenic oomycetes are capable of manipulating and avoiding host immune defences and/or responses.Nevertheless, there are still knowledge gaps to be filled in.
To control phytopathogenic oomycetes, breeders embarked on an ambitious arms race with some oomycetes such as P. infestans more than a hundred years ago but have not been successful, mainly due to its ability to overcome resistance genes.Since the genomics era, RXLR effectors are being exploited by functional genomics strategies to identify resistance genes in host plants, but late blight control is still dependent on the application of chemicals.Also, saprolegniosis control is mostly based on chemical treatment.Therefore, unveiling the infection mechanism(s) and its key players would provide means to develop new sustainable disease control strategies.The development of new gene editing methods is in progress, having the potential to become a powerful tool for functional genomic research in both plant and animal pathogenic oomycetes.
Fig.1e Schematic representation of Oomycota subclasses and its orders.Oomycota species can be found in a variety of ecosystems, and have different lifestyles (saprophytic, pathogenic, or obligate).There are about 600 species in 90 genera, the vast majority being plant pathogenic oomycetes within the orders Rhipidiales, Albuginales, Peronosporales and Pythiales with a few exceptions, while animal pathogenic oomycetes are found mostly in the Saprolegniales order.Figure created using BioRender.com.

Fig. 3 e
Fig. 3 e Schematic representation of selected host-pathogen interactions driven by effectors.Panel A: Plant-Phytophthora interaction.(1) Phytophthora secretes apoplastic effectors like the elicitin INF1 which is recognized by ELR.The ELR-SOBIR1-BAK1 receptor complex formation activates the signalling cascade resulting in PRR-based immunity.(2) In parallel, Phytophthora infestans haustoria secrete cytoplasmic effectors like AVR3a that enter the plant cell.AVR3a stabilizes the plant E3 ligase CMPG1 and negatively regulates INF1-triggered cell death.(3) However, AVR3a can be recognized by the plant resistance protein R3a and activates the NLR-based immunity triggering a hypersensitive response (HR).(4) Another RXLR effector, PsAVH18/PSR1, from P. sojae translocates into the plant nucleus and may interact with DCL1 proteins to disrupt biogenesis of miRNAs that target plant defence related genes; as a consequence the NLR-based immunity can be suppressed.(5) Phytophthora sojae secretes apoplastic hydrolases like the glycoside hydrolase PsXEG1, which upon recognition by RXEG1 initiates the PRR-based immunity.(6) PsXEG1 can also be inhibited by GmGIP1, however, (7) P. sojae secretes the decoy PsXLP1 to protect PsXEG1.(8) In response a second inhibitor GmAP5 degrades PsXEG1, and (9) as a counteract the N-glycosylation protects PsXEG1 from degradation.Panel B: Saprolegnia parasitica secretes effectors that help establish an infection.(1)HTP3, an RxLR-like protein, is recognised by the gp96 receptor, (1a) which normally resides in the ER, but can migrate to the cell membrane (1b) Once bound, it enters the host cell by endocytosis.(4) Inside the cell, HTP3 is release from vesicles with the help of another RxLR-like protein, Htp1.(5) Since HTP3 possess dual nuclease activity it can degrade both DNA and RNA.(3) HTP1 translocates inside host cells in a pathogen independent manner (3a) by binding to tyrosine-O-sulphate present on the cell membrane.(2) SpSSP1, a subtilisin-like serine protease, is secreted into the apoplastic region by the pathogen.It has potentially a role in host immune responses suppression.(6a) The LHS1-like protein of A. invadans is present in the ER (with its cofactors SIL1 and KAR2) and is important for the correct folding of proteins and spore production.(6c) When AiLHS1 is silenced (red cross) A. invadans produces smaller spores with impaired swimming activity which are unable to successfully attach to the host.(6b) Also, point mutations in serine protease genes from A. invadans produced defective zoospores unable to infect fish and induce EUS (epizootic ulcerative syndrome) showing the importance of serine proteases in establishing infection.Figure created using BioRender.com.

Fig. 4 e
Fig. 4 e Summarised representation of host defence mechanisms.Animals and plants respond different when in contact with pathogens.Vertebrates possess a sophisticated immune system, while invertebrates rely only on the innate immune system.Plants, on the other hand, rely mostly on various kinds of immune receptors that recognise effectors from phytopathogenic oomycetes and on a variety of enzymes.Figure partly created using BioRender.com.