Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity

Abstract

The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steadystate levels of non-PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.R protein | NMR | programmed cell death

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

“…This region has a defined and ordered structure corresponding to part of the N-terminal loop region and α 1 and α 2 helices (Fig. 3 A-C), and is clearly distinct from the uptake region in RXLR effectors, which is disordered and flexible (20,33). The internalization region in the flax-rust effector AvrL567 is also completely different in structure to the one in AvrM and corresponds to a region that is partly disordered (residues 27-36) and partly structured (residues 37-50) (24).…”

“…We describe the crystal structure of AvrM and identify functionally important surface regions in the protein, which advances our understanding of the molecular mechanisms underlying how effectors enter host cells and how they are detected by the plant immune system. effector domain of AVR3a from Phytophthora infestans, and not the N-terminal region harboring the RXLR motif, is required for binding to PI3P (19)(20)(21). There is also an ongoing debate in the literature about the significance of the RXLR motifs in host-cell translocation (22,23).…”

Avirulence Protein 3a (AVR3a) from the Potato Pathogen Phytophthora infestans Forms Homodimers through Its Predicted Translocation Region and Does Not Specifically Bind Phospholipids

“…In addition, the authors claimed that RxLR or RxLR-like motifs mediate translocation through binding to phosphoinositol 3-phosphate molecules exposed at the cell surface. However, this view has been recently challenged by two independent studies (4,12). Using recombinant AVR3a from P. infestans, Yaeno et al (4) showed that the effector domain, rather than the RxLR leader, binds to phospholipids (PLPs).…”

“…This model was based on the NMR structure of the Phytophthora capsici homolog, AVR3a4 and shows that the C terminus is flexible and angled away from the four-bundle helical protein core. However, no defined structural information could be obtained from the RxLR leader of P. capsici AVR3a4 to potentially shed light on the requirements for translocation, despite this region being included in the recombinant protein under study (4).…”

Identification of Plasmodiophora brassicae effectors — A challenging goal

“…The motif RxLR, arginine-any amino acid-leucine-arginine, has been identified in the N-terminal of some oomycete and fungal effectors [43,44]. Although the function of the RxLR motif in effector proteins remains unclear, it has been shown to be necessary for translocation into the host cell [45] and to elicit immune responses in plant cells [46].…”