The core effector RipE1 ofRalstonia solanacearuminteracts with and cleaves Exo70B1 and is recognized by the Ptr1 immune receptor

Abstract

Ralstonia solanacearum depends on numerous virulence factors, also known as effectors, to promote disease in a wide range of economically important host-plants. Although some of these effectors have been characterized, none has been shown to target the host secretion machinery so far. Here, a screening was performed, using an extended library of NLR plant immune receptors integrated domains (IDs), to identify new effector targets. The results uncovered that the core effector RipE1, of the R. solanacearum species complex, among other targets, associates with Arabidopsis exocyst component Exo70B1. RipE1, in accordance with its predicted cysteine protease activity, cleaves Exo70B1 in vitro between its sequence and is able to promote Exo70B1 degradation in planta. RipE1 enzymatic activity additionally results in the activation of TN2-dependent cell death. TN2 is an atypical NLR that has been proposed to guard Exo70B1. Despite the fact that RipE1 has been previously reported to activate defense responses in model plant species, we present here a Nicotiana species, in which RipE1 expression does not activate cell death. Overall, this study uncovers a new RipE1 host target, while providing evidence and novel tools to advance in-depth studies of RipE1 and homologues effectors.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1

“…As NbPtr1 is required for the recognition of effectors from different families, probably by monitoring the state of the plasma membrane-associated protein RIN4 (Ahn et al, 2022;Mazo-Molina et al, 2020), we next investigated RipE1 subcellular localization with laser scanning confocal microscopy. Nakano and Mukaihara (2019b) reported a nucleocytoplasmic localization for the N-terminally tagged RipE1 (from strain RS1000), while we and others (Jeon et al, 2020;Tsakiri et al, 2023) observed a plasma membrane localization for RipE1 C-terminally tagged with yellow fluorescent protein (YFP). This difference suggests that RipE1 association with the plasma membrane is mediated through the N-terminal region of the protein.…”

“…The N-terminal region of RipE1 was not predicted to participate in the cysteine-protease fold in a predicted structural model (Tsakiri et al, 2023). This region does not contain a myristylation motif that was reported for several plasma membrane-associated T3S effectors, including AvrRpm1 (Nimchuk et al, 2000).…”

The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity

“…The exocyst complex is a conserved octameric protein complex that regulates the tethering of secretion vesicles to the cell membrane (Mei & Guo, 2019). Being the components of an essential cellular mechanism, exocyst subunits are often targeted by pathogens that manipulate host cell physiology (Michalopoulou et al., 2022; Tsakiri et al., 2022; Wang et al., 2019). Successful plant pathogens use an arsenal of protein effectors, which display a variety of functions (Kotsaridis et al., 2022).…”

From prediction to function: Current practices and challenges towards the functional characterization of type III effectors

“…The AlphaFold database is currently vastly expanding, including additional proteomes and catalogued protein structures for even more accurate predictions. At the publication time, the AlphaFold source code is accessible online (Collabfold) and can be used to predict the protein structure of any protein of interest, as was done for the Ralstonia T3E RipE1 that harbors a cysteine protease domain (Tsakiri et al, 2022). More recently, the algorithm AlphaFold-Multimer has been created that could be used for the modelling of (effector-host protein) interactions (Evans et al, 2021).…”