Proteolytic processing of both RXLR and EER motifs in oomycete effectors

Introduction

Diseases caused by plant pathogens and pests result in a considerable threat to food security, including up to 23% losses of the five most significant food crops (Savary et al., 2019). Amongst the most economically significant disease agents are fungal and oomycete (filamentous) pathogens. The oomycete genus Phytophthora includes some of the most devastating plant pathogens (Kamoun et al., 2015; Derevnina et al., 2016). For example, Phytophthora infestans, causing potato and tomato late blight, precipitated the Irish potato famines of the 19^th century. It remains the most damaging potato and tomato disease globally (Fry et al., 2015; Kamoun et al., 2015).

Phytophthora spp. secrete ‘effector’ proteins that act either outside (apoplastic effectors) or are delivered to the inside (cytoplasmic effectors) of living plant cells. Prominent amongst cytoplasmic effectors are a class containing the conserved Arg-any amino acid-Leu-Arg (RXLR) motif (Rehmany et al., 2005) located closely downstream of the signal peptide. RXLR effectors target multiple proteins and processes at diverse locations inside host cells to suppress immunity (He et al., 2020; Fabro, 2021; Petre et al., 2021; McLellan et al., 2022; Wang et al., 2023).

Many filamentous pathogens, including P. infestans, form haustoria, hyphal infection structures that are intimately associated with living plant cells. Haustoria are sites of cross-kingdom molecular exchange and, as such, represent key battle grounds that determine host susceptibility or resistance (Boevink et al., 2020; Bozkurt & Kamoun, 2020; King et al., 2023). RXLR effectors have been shown to enter plant cells following their unconventional secretion from haustoria. By contrast, although also secreted from haustoria, apoplastic P. infestans effectors follow the canonical ER-to-Golgi pathway that is sensitive to the inhibitor brefeldin A (BFA) (Wang et al., 2017, 2018). Unconventional secretion of cytoplasmic effectors and conventional secretion of apoplastic effectors has also been observed for the fungal pathogen Magnaporthe oryzae (Giraldo et al., 2013). More recently, it has been reported that P. infestans RXLR effectors can be taken into plant host cells via clathrin-mediated endocytosis (CME) (Wang et al., 2023a). Similarly, M. oryzae cytoplasmic effectors have also been observed to enter plant cells via CME (Oliveira-Garcia et al., 2023), hinting at a potential universal strategy employed by haustoria-forming filamentous pathogens (Wang et al., 2023b).

The RXLR motif is required for effector translocation into plant cells (Whisson et al., 2007). However, its precise role has been difficult to elucidate and has often led to controversy (Ellis & Dodds, 2011; Boevink et al., 2020; Bozkurt & Kamoun, 2020). The RXLR motif was reported to bind to phosphoinositide-3-phosphate (PI3P) on the outer surface of plant cells, promoting uptake in a pathogen-autonomous manner (Kale et al., 2010). However, pathogen-independent uptake was drawn into question (Wawra et al., 2013; Wang et al., 2017), as was the PI3P-binding of the RXLR motif (Yaeno et al., 2011; Wawra et al., 2012). Indeed, the RXLR motif has been reported to be a site of proteolytic cleavage before effector secretion (Wawra et al., 2017), implying that it is not responsible for binding to PI3P on the outer surface of the plant cell membrane.

Cleavage at the RXLR motif is reminiscent of proteolytic cleavage at an equivalent motif, RXLXE/D/Q, also called the Plasmodium export element (PEXEL), in effectors of the malaria parasite, which are delivered into host blood cells (Boddey et al., 2010; Russo et al., 2010). As is apparent with the RXLR motif, the PEXEL motif is positionally constrained to within 40 amino acids after the signal peptide (SP) cleavage site (Battacharjee et al., 2006; Win et al., 2007; Win & Kamoun, 2008). Its spatial conservation and rapid proteolytic cleavage, followed by acetylation of the N-terminus, are important for effector secretion via a specialised export pathway (Boddey et al., 2016).

In addition to the RXLR motif, many Phytophthora cytoplasmic effectors also contain a conserved Glu-Glu-Arg (EER) motif, immediately downstream of the RXLR, which has again been implicated in effector delivery into host cells (Whisson et al., 2007). Moreover, a ‘WY’ domain represents a conserved structural fold within the C-terminal half of many RXLR effectors that contributes to host target specificity (Boutemy et al., 2011; Win et al., 2012; Bentham et al., 2023; Li et al., 2023). Interestingly, a number of expressed effectors from Bremia lactucae that are predicted to contain the WY structural fold and are recognised by host resistance proteins only contain the EER motif (Wood et al., 2020). Given that there are functionally characterised effectors containing only the RXLR motif, this raises the possibility that both motifs are effector processing sites (Wang et al., 2023).

In this work, we set out to investigate whether cleavage at the RXLR motif is evident in diverse P. infestans effectors. We discovered that cleavage is indeed evident in effectors containing RXLR-only or RXLR-EER motifs. Moreover, we observed that cleavage occurs after the leucine in the RXLR motif, rather than the second arginine as previously reported (Wawra et al., 2017). We confirm that cleavage of the RXLR is constrained by its location after the SP. Unexpectedly, we discovered that the EER motif is also cleaved after the arginine, revealing that both RXLR and EER are sites for proteolytic processing.