Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense

Maidment, Josephine HR; Franceschetti, Marina; Maqbool, Abbas; Saitoh, Hiromasa; Jantasuriyarat, Chatchawan; Kamoun, Sophien; Terauchi, Ryohei; Banfield, Mark J.

doi:10.1016/j.jbc.2021.100371

Cited by 73 publications

(109 citation statements)

References 75 publications

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“…Studies with the NLR pair Pik from rice have shown that the strength of effector binding to integrated domains in vitro can correlate with immune responses in planta (52–54). Of the AvrRps4 mutants we tested to validate the RRS1 WRKY interface, all except N171A and Q194A prevented binding in vitro (by ITC) and in planta (by co-IP), and these did not give cell death in Nicotiana species when co-expressed with either RRS1-R/RPS4 or RRS1-S/RPS4.…”

Section: Discussionmentioning

confidence: 99%

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Mukhi

Brown

Gorenkin

et al. 2021

Preprint

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Plants use intracellular immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. We report here the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C/RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA binding domain of AtWRKY41, with similar binding affinities. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

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Section: Discussionmentioning

confidence: 99%

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Mukhi

Brown

Gorenkin

et al. 2021

Preprint

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“…To further demonstrate the flexibility of the pOPIN-GG vectors, we next cloned an interaction partner of AVR-PikF, OsHIPP19 (Maidment et al, 2021), into pPGN-C with a 6xHIS-GB1 tag. We co-transformed E. coli SHuffle cells with the 6xHIS-GB1-OsHIPP19 vector and an untagged variant of AVR-PikF (in pPGC-K), expressed the proteins, and performed Ni 2+ -IMAC coupled with size exclusion chromatography (SEC) to purify a complex between AVR-PikF and OsHIPP19 (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

pOPIN-GG: A resource for modular assembly in protein expression vectors

Bentham

Youles

Mendel

et al. 2021

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The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

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“…Next, we tested the influence of the key polymorphisms identified in the yeast-two hybrid mutant screen on in vitro sHMA94 binding. We first measured the effect of the Asp to Asn mutation at position 66 in APikL2, as this is the only polymorphism between APikL2A and APikL2F that is predicted to locate to the HMA binding interface (based on [7,10,46]).…”

Section: Asn-66 Facilitates Apikl2 Binding To Shma94mentioning

confidence: 99%

“…The remarkable finding that a single type of NLR-integrated domain mediates perception of three sequence diverse blast fungus effectors indicates that this pathogen has evolved multiple effectors to target HMA-containing proteins. Indeed, two recent studies validated the view that, before being baited by NLR proteins, AVR-Pik and other HMA binding effectors have convergently evolved to target host proteins of the heavy metal-associated plant proteins (HPPs) and heavy metal-associated isoprenylated plant proteins (HIPPs), collectively referred to as small HMA (sHMA) proteins [45][46][47]. sHMA proteins are metallochaperone-like proteins that belong to an expanded family of up to 100 members in a single grass species that contribute to metal homeostasis and detoxification during abiotic stress [45,48].…”

Section: Introductionmentioning

confidence: 96%

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Bentham

Petit

Win

et al. 2021

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Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

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Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense

Cited by 73 publications

References 75 publications

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

pOPIN-GG: A resource for modular assembly in protein expression vectors

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

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