Piecing together oomycete effector processing and host translocation

Abstract

The gene-for-gene hypothesis, proposed by Flor in 1942, postulates that for each dominant avirulence (Avr) protein in a pathogen, there is a corresponding resistance (R) gene in the host. This interaction triggers host defense mechanisms, such as the hypersensitive response, which effectively limits biotrophic pathogen growth (Flor, 1942). Decades later, the term ‘effector protein’ was introduced to describe proteins secreted or translocated by pathogens to manipulate the host. Understanding how pathogens process and secrete these effectors is crucial for deciphering their pathogenicity.

The zigzag model has been classically used to describe plant immune responses through a dynamic interaction between pathogen-associated molecular patterns (PAMPs) and effectors. This model is readily applied for oomycetes, where plants detect oomycete PAMPs, such as β-1,3- and β-1,6-glucans, or cellulose-binding protein and XEG1 endoglucanase (Judelson & Ah-Fong, 2019) via pattern recognition receptors (PRRs), leading to PAMP-triggered immunity (PTI). In response, oomycetes evolved effectors to suppress PTI, causing effector-triggered susceptibility (ETS), but plants counter this by recognizing specific effectors through R proteins, activating effector-triggered immunity (ETI), which again is thought to be more robust (Jones & Dangl, 2006). Though still holding true for evolution, the zigzag model has been critiqued for oversimplification when it comes to the molecular plant–oomycete interaction. This critique is based on data showing that effector proteins are secreted by oomycetes and sometimes recognized by the host before physical contact even occurs; PTI, ETS and ETI are rather parallel than separate mechanisms.

Despite the crucial role of effector proteins in plant infection, surprisingly little is known about their processing and secretion mechanisms in oomycetes. In other eukaryotic systems, it is well established that conventional secretion follows the classical secretory pathway, where proteins are directed to the endoplasmic reticulum (ER) via an N-terminal signal peptide. From there, proteins are processed and transported to the Golgi apparatus before being secreted out of the cell. Unconventional secretion, on the other hand, bypasses the classical ER–Golgi pathway and involves alternative mechanisms, such as direct translocation across the plasma membrane or via endosomal or vesicular pathways (Wang et al., 2017).

It is expected that some eukaryotic clades have evolved new pathways or variations. In Xu et al. (2025), the cleavage of RXLR and EER motifs in P. infestans effectors provide important insights into how these motifs function in effector processing and secretion. While conventional secretion mechanisms remain central, the possibility of alternative processing routes, influenced by the structural positioning of these motifs, highlights the complex posttranslational modifications that occur before these effectors are delivered into the host plant. In the case of RXLR effectors, the signal peptide (SP) is cleaved off during or after translocation into the ER. This is followed by additional posttranslational modifications, including proteolytic cleavage at the RXLR motif, as shown in Xu et al.'s (2025) study. The general rule is that these effectors follow this conventional pathway, dependent on cellular machinery for proper processing and delivery into the host.

Proteolytic cleavage at the RXLR motif in P. infestans mirrors a similar mechanism seen in other eukaryotic pathogens. The RXLR motif bears a striking resemblance to the RXLXE/D/Q motif, also known as the Plasmodium export element (PEXEL), which is found in malaria parasite effectors. These effectors, like those from P. infestans, are delivered into host cells – in this case, the red blood cells of the human host (Gabriela et al., 2024). Just as the RXLR motif is spatially constrained to within 40 amino acids after the signal peptide cleavage site, so is the PEXEL motif. In Plasmodium, this constraint is crucial for correct cleavage and subsequent effector processing (Gabriela et al., 2024). After cleavage, the N-terminal residue is acetylated, an essential modification for secretion through the specialized export machinery (Boddey et al., 2016). This spatial and functional conservation suggests that both motifs share a fundamental role in ensuring that effectors are processed and secreted correctly into host cells, albeit in different organisms.

In Phytophthora species, many RXLR effectors also contain an adjacent Glu-Glu-Arg (EER) motif, which has similarly been implicated in effector translocation into host plant cells (Whisson et al., 2007). Xu et al. (2025) build upon this by demonstrating for the first time that the EER motif, like the RXLR motif, can also be proteolytically cleaved within Phytophthora (Fig. 1). Their findings challenge previous assumptions that the EER motif is solely involved in host cell delivery, showing instead that it serves as an additional site for processing before secretion. In contrast to Phytophthora, plants lack the necessary molecular machinery to cleave effector proteins at the RXLR or EER motifs. Beyond the RXLR and EER motifs themselves, the structural context of these effectors also plays a critical role in their function. The conserved WY domain, which is commonly found in the C-terminal region of many RXLR effectors, contributes to host target specificity and stability (Bentham et al., 2024; Li et al., 2023). The presence of the WY (Trp-Tyr) domain suggests that effector processing and translocation are closely coordinated with the structural elements that mediate interactions with host plant targets. Interestingly, some effectors from Bremia lactucae – a pathogen of lettuce – contain the WY domain but only the EER motif, raising the possibility that the EER motif alone may serve as a processing site in these proteins (Wood et al., 2020). This further reinforces the idea, as discussed by Wang et al. (2023), that RXLR and EER motifs function as general processing sites in diverse oomycete effectors. Gabriela et al. (2024) provided evidence that in PEXEL proteins, certain amino acids serve dual functions. Mutations in these residues can significantly reduce cleavage, leading to increased retention in the ER and decreased export. In other proteins, similar mutations have a lesser effect, allowing PEXEL proteins to reach the parasitophorous vacuole, though with reduced translocation efficiency. We can learn from the Plasmodium experiments that amino acids within the RXLR and EER motif may contribute differently to the processing and secretion in dependents of the effector function and the amino acid characteristics within the motifs. However, this hypothesis will need further testing, for example, using single point mutations leading to degenerate RXLR motifs such as [KHGQ][X][LMYFIV][RNK] commonly observed in downy mildews (Wood et al., 2020).

Xu et al. (2025) expand our understanding by providing experimental evidence that proteolytic cleavage at both the RXLR and EER motifs is a widespread phenomenon. This not only supports the idea of functional conservation across oomycete pathogens, but also opens avenues for exploring how similar motifs may be processed in other eukaryotic pathogens, such as Plasmodium, highlighting evolutionary parallels in effector secretion mechanisms. Furthermore, it raises questions about the evolution and conservation of secretory pathways in pathogens in the context of their host infection strategies, given that not all oomycetes possess RXLR(-like) effectors. The discovery of dual cleavage sites adds complexity to our understanding of effector biology in P. infestans. Combined with recent findings on effector uptake via endocytosis (Oliveira-Garcia et al., 2023; Wang et al., 2023), this research points toward an exciting direction in fully understanding how these essential proteins are delivered in plant–oomycete interactions and beyond.