Two folds, many faces: The Magnaporthe oryzae MAX effector AVR-Pia targets novel rice HMA domain-containing proteins

Josephine H. R. Maidment; Svenja C. Saile; Aurélien Bocquet; Céline Thivolle; Léo Bourcet; Lisa-Fatimatou Planel; Muriel Gelin; Thomas Kroj; André Padilla; Karine de Guillen; Stella Cesari

doi:10.1371/journal.ppat.1014382

Abstract

Phytopathogenic fungi secrete effector proteins to promote virulence. The MAX (Magnaporthe Avrs and ToxB-like) effectors form a structurally conserved family despite significant sequence diversity. AVR-Pia, a MAX effector from the rice blast fungus Magnaporthe oryzae, is recognised by the model rice nucleotide-binding leucine-rich repeat (NLR) receptor pair OsRGA4/OsRGA5 via direct binding to a heavy metal-associated (HMA) integrated domain (ID) in OsRGA5. While the structural basis of AVR-Pia recognition is well defined, the role of this effector in promoting virulence has remained elusive. Here, we reveal that AVR-Pia specifically interacts with four previously uncharacterised rice HMA domain-containing proteins, three HMA Plant Proteins (OsHPP09, OsHPP10 and OsHPP11), and one HMA Isoprenylated Plant Protein (OsHIPP21). AVR-Pia binds these proteins in vitro and in planta, engaging their HMA domains with differential affinities. Notably, AVR-Pia binds OsHPP09-HMA with considerably higher affinity than the HMA-ID of OsRGA5. By solving the crystal structure of the AVR-Pia/OsHPP09-HMA complex, we identified additional molecular contacts at the interface which underpin high affinity binding. Importantly, the H(I)PPs identified as AVR-Pia interactors are distinct from those bound by the MAX effectors AVR-Pik and Pwl2, underscoring target specialisation within the MAX effector family. Further, structural analyses of the AVR-Pia/OsHPP09-HMA complex revealed a markedly different interface compared to other MAX effector/H(I)PP complexes. Finally, structure-guided mutagenesis of OsHPP09 identified a single residue that is critical for AVR-Pia binding. This work provides structural insight into how distinct MAX effectors exploit HMA domain-containing proteins and offers a foundation towards targeted modification of HMA domains to disrupt effector binding and enhance cereal resistance to blast disease.

Author summary

Blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the world’s most devastating rice diseases. During infection, M. oryzae secretes a diverse set of molecules, known as effectors, that manipulate host factors to promote disease. The MAX (Magnaporthe AVRs and ToxB-like) family of effectors are structurally conserved but highly diverse in sequence, and form an important part of this virulence arsenal. Certain effectors are recognised by plant immune receptors to activate defences. The MAX effector AVR-Pia is detected by the model immune receptor pair OsRGA4/OsRGA5 in certain rice varieties, conferring resistance to blast, and the molecular detail of this recognition is well-defined. However, the host proteins targeted by AVR-Pia to promote virulence have remained unknown. Here, we identify specific members of an expanded family of HMA domain-containing proteins as direct interactors of AVR-Pia. Combining structural and biochemical analyses, we uncover the molecular details underpinning AVR-Pia/HMA domain binding. Further, we highlight that distinct MAX effectors in M. oryzae target specific sets of HMA domain-containing proteins at different interfaces. Importantly, our structural insights provide a foundation for engineering HMA domains that resist effector binding, offering potential strategies to develop rice and other cereals with improved resistance to blast disease.

Citation: Maidment JHR, Saile SC, Bocquet A, Thivolle C, Bourcet L, Planel L-F, et al. (2026) Two folds, many faces: The Magnaporthe oryzae MAX effector AVR-Pia targets novel rice HMA domain-containing proteins. PLoS Pathog 22(7): e1014382. https://doi.org/10.1371/journal.ppat.1014382

Editor: Ksenia Krasileva, University of California Berkeley, UNITED STATES OF AMERICA

Received: December 1, 2025; Accepted: June 15, 2026; Published: July 13, 2026

Copyright: © 2026 Maidment et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The protein structure of the complex between OsHPP09-HMA and AVR-Pia has been deposited in the Protein Data Bank with accession number 9RSV (pdb_00009RSV). X-ray diffraction data are publicly available via the European Synchrotron Radiation Facility (https://doi.esrf.fr/10.15151/ESRF-DC-2199982133) [94]. Raw data, including uncropped images of SDS-PAGE gels, Western blots and yeast two-hybrid assays, as well as the AlphaFold2/3 models described in this work, are available on Zenodo (DOI:10.5281/zenodo.20845674).

Funding: SC was awarded a HORIZON EUROPE ERC-2029-STG (grant number: 852482-ii-MAX) from the European Research Council (https://erc.europa.eu/homepage). SCS was awarded a Walter Benjamin Fellowship (https://www.dfg.de/en/research-funding/funding-opportunities/programmes/individual/walter-benjamin) from the German Research Foundation (DFG, project number 550008416). This work used the NMR and RX facilities of the Integrated Biophysics and Structural Biology Platform (PIBBS) of the CBS (http://www.cbs.cnrs.fr/index.php/fr/plateformes-equipements/pibbs). PIBBS is a GIS-IBISA platform and belongs to the French Infrastructure for Integrated Structural Biology (FRISBI), supported by the National Research Agency (ANR-10-INBS-05). The funders did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The fungal pathogen Magnaporthe oryzae (syn. Pyricularia oryzae) is the causative agent of rice blast, one of the most devastating diseases affecting cultivated rice worldwide [1,2]. M. oryzae also causes severe yield losses in other cereals, including millets, barley and wheat, threatening global food security [1].

To successfully colonise host plants, M. oryzae must evade plant defence responses and manipulate host cellular pathways to its advantage. Central to infection is the secretion of a large repertoire of effector proteins, many of which can be categorised into structural families [3]. In M. oryzae, the Magnaporthe AVRs and ToxB-like (MAX) effectors form the largest structural family [4]. MAX effectors are defined by a conserved fold consisting of two β-sheets, each composed of three antiparallel β-strands and stabilised by one or more disulphide bonds. Despite this shared structure, there is little amino acid sequence similarity between MAX effectors. They differ in surface properties and some carry variable N- and C-terminal extensions, which can influence their folding pathway [5] and mediate interactions with host proteins [3,6–9].

AVR-Pia is a MAX effector present in approximately 20% of rice-infecting M. oryzae isolates. It is highly expressed during the biotrophic phase of host colonisation and contributes to virulence on rice [4,7,10]. AVR-Pia can be recognised by the intracellular immune receptor OsRGA5, a nucleotide-binding leucine-rich repeat (NLR) protein that functions together with the helper NLR OsRGA4 to activate immune responses and restrict the spread of M. oryzae [11–13]. Canonical plant NLRs consist of an N-terminal signalling domain, a central nucleotide-binding domain and a C-terminal leucine-rich repeat domain [14]. However, many NLRs also carry non-canonical integrated domains (IDs), thought to originate from host proteins targeted by pathogen effectors [15]. These IDs, found across diverse plant species, are believed to function as mimics or baits for effector recognition (“integrated decoy” model) [15–18]. Despite sharing limited sequence similarity, both AVR-Pia and a second MAX effector, AVR1-CO39, bind to the heavy metal-associated (HMA) ID of OsRGA5 [11,12]. While this suggests that these effectors may target host proteins containing HMA domains to promote disease, no host targets of AVR-Pia or AVR1-CO39 have been identified, and their specific virulence functions remain unknown.

The OsRGA5 HMA ID shares sequence similarity with a family of HMA domain-containing metallochaperone-like proteins. The family can be subdivided into two groups: HMA domain-containing Isoprenylated Plant Proteins (HIPPs) that carry a C-terminal CaaX isoprenylation motif, and HMA domain-containing Plant Proteins (HPPs) that lack this motif [19,20]. The two groups are collectively referred to hereafter as H(I)PPs. Isoprenylation, a post-translational modification involving the covalent attachment of an isoprenoid lipid to the cysteine residue in the CaaX motif, has been reported to facilitate membrane association [21]. Many HIPPs harbour proline-rich regions between the HMA domain and the CaaX motif, potentially mediating protein-protein interactions [19,22]. Typically, HMA domains feature a metal-binding MxCxxC motif, implicating H(I)PPs in roles related to metal homeostasis [23,24]. However, approximately a third of rice H(I)PPs lack one or both cysteines in their MxCxxC motif, suggesting functional diversification beyond metal binding. H(I)PPs have undergone extensive expansion in vascular plants, with approximately 100 members identified in rice [19,25–27]. Most remain functionally uncharacterized, although several have been implicated in response to various biotic and abiotic stresses [19,28–30]. Increasing evidence points to a broader role of H(I)PPs as susceptibility factors in interactions with pathogens, pests, and viruses [27,31–41].

In cereals, H(I)PPs have been identified as susceptibility factors for M. oryzae infection [27,33,34]. A loss-of-function allele of OsHIPP05 (Pi21) contributes to blast resistance in the field [33]. OsHIPP20 is also a susceptibility gene; a loss-of-function mutation reduces the susceptibility of rice to M. oryzae [27]. Some H(I)PPs are targeted by M. oryzae MAX effectors AVR-Pik and APikL2 [8,27,42]. AVR-Pik specifically interacts with members of a phylogenetic subclade of OsH(I)PPs, referred to as clade A, including OsHIPP19, OsHIPP20, OsHPP03, and OsHPP04 [8,27]. The paired rice NLR proteins OsPik-1 and OsPik-2 mediate recognition of AVR-Pik, which requires direct binding of the effector to an HMA ID in OsPik-1 [43–45]. The HMA domain of OsPik-1 belongs to the same phylogenetic clade as the H(I)PPs targeted by AVR-Pik, reinforcing the idea that effector targets may serve as templates for domain integration into NLRs.

Structural analyses of complexes between AVR-PikF or APikL2 and the HMA domains of their HIPP targets or cognate NLR receptors have revealed a conserved binding interface centring on β3 of the MAX fold and β4 of the HMA domain [8,42,45,46]. By contrast, the crystal structure of AVR1-CO39 bound to the HMA ID of OsRGA5 revealed a markedly different interface involving β2 of both AVR1-CO39 and OsRGA5-HMA [12]. NMR titration experiments, structural modelling, and mutagenesis assays indicated that AVR-Pia binds OsRGA5-HMA at a similar interface [12,13,47]. Additionally, the crystal structure of AVR-Pia in complex with OsPikp-1-HMA revealed a comparable binding interface, indicating that AVR-Pia binds different HMA domains in a structurally similar manner [48].The crystal structure of the MAX effector Pwl2 bound to OsHIPP43 also revealed an interface involving β2 of the Pwl2 MAX fold and β2 of OsHIPP43-HMA [9]. However, unlike AVR-Pia and AVR1-CO39, Pwl2 has a C-terminal extension, comprising an ⍺-helix and an unstructured region, which contributes significantly to HMA binding [9]. Collectively, these structural studies underscore the remarkable diversity with which MAX effectors have evolved to bind various HMA domain-containing proteins. They support the hypothesis that AVR-Pia and AVR1-CO39 target distinct H(I)PPs through interfaces that differ from those employed by other MAX effectors.

Here, we identified four OsH(I)PPs as specific interactors of AVR-Pia, none of which are bound by the MAX effectors AVR1-CO39, AVR-Pik and Pwl2. Using biochemical and biophysical approaches, we characterised the interaction between AVR-Pia and one of the interactors, OsHPP09. The crystal structure of AVR-Pia/OsHPP09-HMA complex showed global similarity to the AVR1-CO39/OsRGA5-HMA complex but revealed more extensive intermolecular hydrogen bonding. Guided by the AVR-Pia/OsHPP09-HMA structure, we identified a point mutation in OsHPP09 which disrupts its interaction with AVR-Pia without compromising the HMA fold.

Results

AVR-Pia interacts with rice H(I)PPs in Y2H screening

As AVR-Pia is recognised by OsRGA5 through direct binding to its HMA ID [11,13], we hypothesised that AVR-Pia may target HMA domain-containing proteins to promote host susceptibility. We conducted pairwise yeast two-hybrid (Y2H) assays, using a signal peptide-deleted version of AVR-Pia (dSP-AVR-Pia, 20–85 aa) fused to the GAL4 activation domain (AD) against a rice HMA domain library [49] comprising 78 rice H(I)PP HMA domains fused to the GAL4 DNA binding domain (BD). Five HMA domains interacted with AVR-Pia: OsHPP09 (LOC_Os03g02070), OsHIPP41 (LOC_Os03g06080), OsHPP11 (LOC_Os04g45130), OsHIPP14 (LOC_Os04g39320) and OsHIPP39 (LOC_Os03g02860) (Figs 1a and S1–S2 Fig and S1 Table). When paired with AVR-Pia, OsHPP09-HMA conferred strong yeast growth on interaction-selective media, indicating a robust interaction, while yeast growth for the remaining candidates was weaker.

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Fig 1. AVR-Pia interacts with OsH(I)PPs in yeast two-hybrid, in planta and in vitro.

a Y2H assay of AVR-Pia and MAX58 (both without signal peptides, ∆SP) with either the HMA domain of OsH(I)PPs or full-length (FL) proteins. AVR-Pia/OsRGA5_Cter (883-1116 aa) and AVR-Pia/OsHPP09 served as positive controls (upper and lower panels, respectively). Serial dilutions of diploid yeast clones were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW), or to assess protein-protein interactions (SD/-LWH and SD/-LWH supplemented with 0.5 mM 3-amino-1,2,4-triazole (3AT)). Photos were taken after 7 days of incubation. Ø indicates positions where no yeast was spotted. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. b Split luciferase complementation assay of AVR-Pia and MAX58 (both ∆SP; fused to the N-terminal part of luciferase; 3xHA:NLuc:MAX) with FL OsH(I)PPs (fused to the C-terminal part of luciferase: 3xFlag:CLuc:OsH(I)PP). Indicated constructs were transiently co-expressed in N. benthamiana leaves and leaf discs were harvested 2 days post-infiltration for luminescence measurements. AVR-Pia/OsRGA5_Cter (883-1116 aa) was used as a positive control, and leaves infiltrated with P19 alone as background (BG) control. Box plots show the median (line), mean (cross) and upper/lower quartiles (box limits), whiskers extend to the most extreme data points within 1.5x the interquartile range, with outliers plotted individually. Three independent replicates were performed (n = 8 per replicate) except for AVR-Pia/OsHPP09: n = 16 in replicates 1 and 2. Different letters indicate statistically significant differences based on a pairwise Wilcoxon test (α = 0.05). c Analytical gel filtration traces obtained from injection of OsHPP09-HMA alone (top panel), AVR-Pia alone (middle panel) and the two proteins in a 1:1 molar ratio (bottom panel). Significant peaks are indicated by dashed lines with the elution volume labelled. SDS-PAGE gel inserts show fractions from the peak elution volumes indicated by the grey shaded regions. OsHPP09-HMA absorbs light at 280 nm poorly (molar extinction coefficient of 1490 M^-1 cm^-1) so the peak corresponding to OsHPP09-HMA is small. The elution volume observed for OsHPP09-HMA is consistent with dimerization, as has been observed for other purified HMA domains [12,45].

https://doi.org/10.1371/journal.ppat.1014382.g001

We additionally tested the HMA library against the well-characterised MAX effectors Pwl2 and AVR-PikD. Consistent with earlier publications, we observed dSP-Pwl2 (22–145 aa) interacting with the HMA domain of OsHIPP43, and dSP-AVR-PikD (22–113 aa) interacting with the HMA domains of OsHIPP19, OsHIPP20, and other phylogenetically related HMA domains [8,9,27,34] (S1 and S2 Figs), confirming the sensitivity of our screen.

In parallel, we performed an independent Y2H screen using a rice cDNA library. This identified OsHIPP21 (LOC_Os09g09830) as a putative interactor of AVR-Pia (S1 and S2 Tables).

For each interacting HMA domain, we next tested whether full-length (FL) proteins also bind AVR-Pia in pairwise Y2H assays. Interestingly, interaction between AVR-Pia and FL OsH(I)PP proteins was only observed for OsHPP09, OsHPP11 and OsHIPP21 (Figs 1a and S3), despite confirmed expression of all proteins (S4a, S4b, S4c Fig). To exclude the possibility that the isoprenylation (CaaX) motif of studied FL HIPPs affects nuclear localisation in yeast [21], we mutated the cysteine to serine. However, OsHIPP14^C187S, OsHIPP39^C190S and OsHIPP41^C152S did not interact with AVR-Pia in Y2H assays (S3 and S4a, S4b, S4c Figs).

The HMA domains of OsHPP09 and OsHPP11 share 91.5% and 74.6% amino acid sequence identity with OsHPP10 (LOC_Os10g36200) (S5 Fig). Surprisingly, AVR-Pia did not interact with OsHPP10-HMA nor the FL protein in Y2H assays (Fig 1a). By contrast, OsHIPP21-HMA shares only 30–40% sequence identity with the HMA domains of the three OsHPPs (S5 Fig), and while the HMA domain of OsHIPP21 did not interact with AVR-Pia, FL OsHIPP21 did. Taken together, these results suggest that AVR-Pia binds and potentially targets OsHPP09, OsHPP11, and OsHIPP21.

Previous work classified MAX effectors into 20 structural groups [6]. MAX58 belongs to the same group as AVR-Pia, but differs in surface properties [6]. dSP-MAX58 (20–86 aa) did not interact with any of the tested OsH(I)PPs in Y2H assays (Figs 1a and S1), although the effector protein was expressed (S4c Fig), suggesting that, despite their shared structural fold, effectors with distinct surface properties target different host proteins.

AVR-Pia interacts with rice H(I)PPs in planta

To test whether AVR-Pia and the four OsH(I)PPs interact in planta, we performed split-luciferase complementation assays in Nicotiana benthamiana. 3xFlag-CLuc-OsH(I)PP FL proteins were co-expressed with 3xHA-NLuc-dSP-AVR-Pia or 3xHA-NLuc-dSP-MAX58. Co-expression of FL OsHPP09, OsHPP11, and OsHIPP21 with AVR-Pia led to high luminescence levels, exceeding those observed for the positive control OsRGA5_Cter (883–1116 aa, including the HMA ID) (Fig 1b). Strikingly, a strong luminescence signal was observed for OsHPP10/AVR-Pia, within a similar range to OsHPP09 and OsHPP11 and exceeding OsHIPP21 (Fig 1b). Consistent with the Y2H results, no interaction was observed in planta between AVR-Pia and other tested FL OsHIPPs, including OsHIPP14, OsHIPP39 and OsHIPP41 (Fig 1b). MAX58 showed no interaction with any of the tested OsH(I)PPs (Fig 1b). Expression of all proteins was confirmed by immunoblot analyses (S6 Fig). These data demonstrate that AVR-Pia interacts in planta with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21.

AlphaFold Multimer models cannot differentiate between HMA domains which are bound by AVR-Pia and those which are not

Several studies have used AlphaFold Multimer to distinguish interacting from non-interacting pairs of effectors/host proteins [27,50,51]. We therefore tested whether structural models of AVR-Pia in complex with experimentally verified interactors (OsHPP09-HMA, OsHPP10-HMA, OsHPP11-HMA and OsHIPP21-HMA) showed differences, either in the models themselves or in the associated confidence metrics, compared to structural models of AVR-Pia with non-interacting HMA domains. We modelled AVR-Pia/HMA complexes using AlphaFold2 [52–54] and AlphaFold3 [55]. High confidence models were obtained for all AVR-Pia/HMA pairs except AVR-Pia/OsHIPP42-HMA using AlphaFold2 (S7 Fig) and AVR-Pia/OsHIPP41-HMA using AlphaFold3 (S8 Fig). For the AlphaFold2 models, interface analysis with qtPISA revealed a tendency towards increased interface area for AVR-Pia-interacting HMA domains compared to non-interacting HMA domains. (S7 Fig). By contrast, there were no obvious differences for AlphaFold3 models (S8 Fig). Overall, AlphaFold could not clearly differentiate between HMA domains bound or not bound by AVR-Pia.

AVR-Pia interacts with the HMA domains of OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 in vitro with varying affinities

Recombinant AVR-Pia and HMA domains were produced and purified from E. coli (S9–S11 Figs). We first tested for interaction between purified OsHPP09-HMA and AVR-Pia using analytical size exclusion chromatography. When analysed alone, the peak elution volumes for AVR-Pia and OsHPP09-HMA were 15.3 ml and 13.0 ml, respectively (Fig 1c). The elution volume of OsHPP09-HMA is consistent with dimerization, as has been reported for the integrated HMA domains of the NLR proteins OsPikp-1 and OsRGA5 in solution [12,45]. Modelling an OsHPP09-HMA dimer with AlphaFold3 predicts a similar dimerization interface formed from backbone hydrogen bonding between the β2 strands of the two protomers, which are arranged in opposing directions such that a continuous antiparallel β-sheet is formed from the eight β-strands of the two OsHPP09-HMA domains (S12 Fig). This dimerization interface overlaps with the AVR-Pia binding surface predicted by AlphaFold. Following incubation with OsHPP09-HMA (1:1 effector:HMA ratio), the peak elution volume of AVR-Pia shifted to 12.7 ml. This is consistent with disruption of the OsHPP09-HMA dimer and formation of a 1:1 AVR-Pia/OsHPP09-HMA complex (Fig 1c). The co-elution of the two proteins was supported by SDS-PAGE analysis of elution fractions (Fig 1c).

To determine the affinity with which AVR-Pia binds to the HMA domains of OsHPP09, OsHPP10, OsHPP11 and OsHIPP21, we used isothermal titration calorimetry (ITC). Titration of AVR-Pia into a solution containing any of the purified HMA domains resulted in negative peaks in the titration curve (Figs 2 and S13), indicating an exothermic binding interaction. Dissociation constant (K_D) values were determined from binding isotherms (Figs 2 and S13) fitted to a single site model. AVR-Pia bound to OsHPP09-HMA with an apparent K_D of 115 nM (Fig 2). Despite differing in just six amino acids, AVR-Pia bound OsHPP10-HMA with significantly lower affinity, with an apparent K_D of 5.26 µM. The binding affinities of AVR-Pia and OsHPP11-HMA or OsHIPP21-HMA fell between these two extremes, with apparent K_Ds of 772 nM and 1.29 µM, respectively (Figs 2 and S13–S15 and S3 Table). The apparent K_Ds, determined by ITC, for the interactions between OsRGA5-HMA and AVR-Pia or AVR1-CO39 were previously reported as 7.8 µM [13] and 5.1 µM [12], respectively.

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Fig 2. AVR-Pia binds the HMA domains of OsH(I)PPs with varying affinities.

a Representative ITC experiments for each of the AVR-Pia/HMA interactions tested. Top panel shows the raw thermogram obtained from titration of AVR-Pia into a solution containing the purified HMA domains. Central panel shows the integrated heats (coloured dots) and binding isotherm fitted to a single site model (black line) using the MicroCal PEAQ-ITC analysis software (Malvern Panalytical). Bottom panel shows the differences (coloured crosses) between the modelled and observed values (residuals). b Dot plot representation of the apparent K_D for each of the AVR-Pia/HMA interactions in three independent experiments. Coloured dots represent the K_D values determined in each replicate with coloured lines representing the error associated with the K_D value as determined by the MicroCal PEAQ-ITC analysis software (Malvern Panalytical).

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AVR-Pia-H3 does not interact with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21

Some rice-infecting M. oryzae isolates carry an allele of AVR-Pia, AVR-Pia-H3, which contains two non-synonymous polymorphisms (F24S and T46N) [11] (S16 Fig). These polymorphisms interfere with OsRGA5-HMA binding and allow evasion of OsRGA4/OsRGA5-mediated immunity [11,13]. Pairwise Y2H assays revealed that AVR-Pia-H3 failed to interact with FL OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 or their HMA domains (Fig 3a). In planta split-luciferase complementation assays gave only weak luminescence signals for AVR-Pia-H3 with OsH(I)PPs, comparable to the negative control pairs OsRGA5_Cter/AVR-Pia-H3 and OsHPP09/MAX58, and markedly lower than the positive control OsHPP09/AVR-Pia (Fig 3b). Protein expression in yeast and in planta was confirmed by immunoblot (S4c and S17 Figs). Analysis of a 1:1 effector:HMA mixture of purified recombinant AVR-Pia-H3 and OsHPP09-HMA by analytical size exclusion chromatography showed no co-elution of the two proteins, indicating that they did not form a stable complex in vitro (Fig 3c).

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Fig 3. AVR-Pia-H3 does not interact with OsHPP09, OsHPP10, OsHPP11 and HIPP21.

a Y2H interaction analysis between AVR-Pia-H3 (∆SP) and either the HMA domain of OsH(I)PPs or full-length (FL) proteins. OsHPP09/AVR-Pia served as positive control. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH). Photos were taken after 7 days of incubation. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. b Split luciferase complementation assay of AVR-Pia-H3 (∆SP; fused to the N-terminal part of luciferase; 3xHA:NLuc:MAX) with FL OsH(I)PPs (fused to the C-terminal part of luciferase: 3xFlag:CLuc:OsH(I)PP). Indicated constructs were transiently co-expressed in N. benthamiana leaves and leaf discs were harvested 2 days post-infiltration for luminescence measurements. AVR-Pia/OsHPP09 served as a positive control, MAX58/OsHPP09 and AVR-Pia-H3/OsRGA5_Cter (883-1116 aa) as negative controls, and leaves infiltrated with P19 alone as background (BG) control. Box plots show the median (line), mean (cross) and upper/lower quartiles (box limits), whiskers extend to the most extreme data points within 1.5x the interquartile range, with outliers plotted individually. Three independent replicates were performed, with n = 8 per combination per replicate. Due to experimental constraints, for AVR-Pia/OsHPP09, n = 16 in replicates 1 and 2. Data points are shown with shapes indicating the replicate. Different letters indicate statistically significant differences based on a pairwise Wilcoxon test (α = 0.05). c Analytical gel filtration traces obtained from injection of OsHPP09-HMA alone (top panel), AVR-Pia-H3 alone (middle panel) and the proteins combined (1:1 molar ratio; bottom panel). Significant peaks are indicated by coloured dashed lines with elution volume labelled. SDS-PAGE gel inserts show fractions from peak elution volumes indicated by grey shaded regions. OsHPP09-HMA absorbs light at 280 nm poorly (molar extinction coefficient: 1490 M^-1 cm^-1) so the peak corresponding to OsHPP09-HMA is small.

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Together, these data indicate that the F24S and T46N polymorphisms that disrupt binding of AVR-Pia to the HMA ID of OsRGA5 also interfere with binding to its putative host targets.

AVR1-CO39 does not interact with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21

As the HMA ID of OsRGA5 also binds the MAX effector AVR1-CO39 [11,12], we tested whether AVR1-CO39 binds to the same H(I)PPs as AVR-Pia. dSP-AVR1-CO39 (22–89 aa) did not interact with FL OsHPP09, OsHPP10, OsHPP11 or OsHIPP21, nor their HMA domains, in Y2H, whereas interaction with OsRGA5_Cter was confirmed (Fig 4a). Effector expression was confirmed by immunoblot (S4c Fig).

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Fig 4. OsH(I)PPs interacting with AVR-Pia do not interact with AVR1-CO39.

a Y2H interaction analysis between AVR1-CO39 (∆SP) and either the HMA domain of OsH(I)PPs or full-length (FL) proteins. AVR1-CO39/OsRGA5_Cter (883-1116 aa) served as a positive control. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH). Photos were taken after 7 days of incubation. Ø indicates positions where no yeast was spotted. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. b Split-luciferase complementation assay of ∆SP AVR1-CO39 with FL OsH(I)PPs and OsRGA5_Cter (883-1116 aa). MAX effectors were fused to the N-terminal parts of luciferase (3xHA:NLuc:MAX), while OsH(I)PP FL proteins and OsRGA5_Cter were fused to its C-terminal part (3xFlag:CLuc:OsH(I)PP/OsRGA5_Cter). Constructs were transiently co-expressed in N. benthamiana leaves and leaf discs were harvested 2 days post-infiltration for luminescence measurements. AVR-Pia or AVR1-CO39 with OsPi21 served as negative controls and AVR-Pia/OsHPP09 as a positive control. Box plots show the median (line), mean (cross) and upper/lower quartiles (box limits), whiskers extend to the most extreme data points within 1.5x the interquartile range, with outliers plotted individually. Three independent replicates were performed, with n = 8 per combination per replicate. All data points are shown with shapes indicating the replicate. Different letters indicate statistically significant differences based on a pairwise Wilcoxon test (α = 0.05). c Analytical gel filtration traces obtained from injection of OsHPP09-HMA alone (top panel), AVR1-CO39 alone (middle panel) and the proteins combined (1:1 molar ratio; bottom panel). Significant peaks are indicated by coloured dashed lines with elution volume labelled. SDS-PAGE gel inserts show fractions from peak elution volumes indicated by grey shaded regions. The small peak at ~13.7 ml for AVR1-CO39 is likely due to dimerisation of the AVR1-CO39 protein; the quantity of the dimer is insufficient to observe a band on the SDS-PAGE gel. OsHPP09-HMA absorbs light at 280 nm poorly (molar extinction coefficient: 1490 M^-1 cm^-1) so the peak corresponding to OsHPP09-HMA is small.

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In split-luciferase complementation assays, we observed weak luminescence for AVR1-CO39 with FL OsHPP09, OsHPP10 and OsHPP11, similar to the luminescence observed with OsRGA5_Cter but substantially lower than the level observed for AVR-Pia/OsHPP09 (Fig 4b). Consistent with the Y2H results, no interaction was detected between FL OsHIPP21 and AVR1-CO39, with similar luminescence to the negative control OsPi21/AVR1-CO39 (Fig 4b). Protein expression was validated by immunoblot (S18 Fig). Analytical size-exclusion chromatography with purified proteins further supported the absence of complex formation between OsHPP09-HMA and AVR1-CO39 (Fig 4c).

Collectively, these results suggest that, while AVR1-CO39 and AVR-Pia are both recognised by the same HMA ID in OsRGA5, they do not bind the same H(I)PPs.

AVR-Pia interacts with a distinct group of H(I)PPs to AVR-Pik and Pwl2

Previous studies have shown that the MAX effectors AVR-PikC, AVR-PikD and Pwl2 target H(I)PPs [8,9,27,34]. However, dSP-AVR-PikC (22–113 aa), dSP-AVR-PikD (22–113 aa) and dSP-Pwl2 (22–145 aa) did not bind to the AVR-Pia interactors OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 in Y2H assays (Fig 5a), while we confirmed the known interactions of AVR-PikC/AVR-PikD with OsHIPP19/OsHIPP20, and Pwl2 with OsHIPP43 (Fig 5a). Interestingly, AVR-Pia interacted with FL OsHIPP19 under low-stringency Y2H conditions (Fig 5a).

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Fig 5. OsH(I)PPs interacting with AVR-Pia do not interact with AVR-Pik or Pwl2.

a Y2H interaction analysis between AVR-PikC, AVR-PikD, Pwl2 and AVR-Pia (all ∆SP) and either the HMA domain of OsH(I)PPs or full-length (FL) proteins. OsHIPP19/AVR-PikC, OsHIPP20/AVR-PikC, OsHIPP19/AVR-PikD, OsHIPP20/AVR-PikD and OsHIPP43/Pwl2 served as positive controls. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH). Photos were taken after 7 days of incubation. AD, activating domain; BD, binding domain. b Split luciferase complementation assay of MAX effectors (∆SP; fused to the N-terminal part of luciferase: 3xHA:NLuc:MAX) with OsH(I)PP FL proteins (fused to the C-terminal part of luciferase: 3xFlag:CLuc:OsH(I)PP). AVR-PikC/AVR-PikD with OsHIPP19/20 and Pwl2 with OsHIPP43 served as positive controls, AVR-Pia with OsHIPP43 served as a negative control. Leaves infiltrated with P19 alone served as background (BG) control. Box plots show the median (line), mean (cross) and upper/lower quartiles (box limits), whiskers extend to the most extreme data points within 1.5x the interquartile range, with outliers plotted individually Three independent replicates were performed, with n = 8 per combination per replicate, except for AVR-Pia/OsHIPP19 and AVR-Pia/OsHIPP20 in AVR-PikC and AVR-PikD panels: n = 4 in replicates 1 and 3. For BG control in AVR-PikC and AVR-PikD panels: n = 3 in replicates 1 and 3 and for BG control in Pwl2 panel: n = 2 in replicates 1 and 3. For AVR-PikD/OsHIPP19 n = 16 in replicates 1 and 3 and n = 24 in replicate 2. Data points are shown with shapes indicating the replicate. Different letters indicate statistically significant differences based on a pairwise Wilcoxon test (α = 0.05).

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The lack of interaction between AVR-PikC and AVR-PikD with FL OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 was validated through split-luciferase complementation assays (Fig 5b). AVR-Pia showed an interaction with OsHIPP19 similar to that observed for AVR-PikD, and a weaker interaction with OsHIPP20 (Fig 5b). Co-expression of Pwl2 with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 resulted in very weak luminescence. Protein expression was confirmed by immunoblot (S19 Fig).

Together, our findings indicate that the HMA domains bound by AVR-Pia are not targeted by AVR-Pik and Pwl2, suggesting that despite sharing a conserved MAX effector fold, these effectors target different H(I)PPs.

AVR-Pia interacts with the four HMA domains through a similar surface

To determine which residues of AVR-Pia are involved in HMA binding, NMR titration with ¹H-¹⁵N-HSQC NMR 2D spectra of ¹⁵N-labelled AVR-Pia alone and in the presence of unlabelled OsHPP09-HMA were performed. HSQC spectra for ¹⁵N-labelled AVR-Pia had previously been collected, and the resonances of amide cross-peaks had been assigned [4]. Binding of AVR-Pia to OsHPP09-HMA alters the chemical environment of the amino acids located at the binding interface, resulting in a change in the chemical shift observed for those amino acids in the ¹⁵N HSQC spectra. The ¹⁵N HSQC spectra of the complex AVR-Pia with unlabelled OsHPP09-HMA were reassigned using ¹⁵N,¹³C-AVR-Pia and two- and three-dimensional NMR experiments (S20 Fig). Depending on the exchange rate constant (kex) between the bound and unbound states and on the frequency difference (∆ω) between the corresponding resonances in these two states, different NMR exchange regimes occur. NMR titration showed that the AVR-Pia:OsHPP09-HMA complex was in slow exchange with kex <<∆ω since separate resonances appeared for individual species (S21 Fig). An NMR slow exchange regime indicates a relatively high affinity (<0.1 µM) between the two proteins. The chemical shift perturbations of AVR-Pia in the absence and presence of OsHPP09-HMA were calculated, and significant changes in chemical shift (> + 1σ) were observed for multiple residues in β2 and β3 as well as R23 from β1 of AVR-Pia, consistent with the interface modelled by AlphaFold2 and AlphaFold3 (S22 Fig).

¹H-¹⁵N-HSQC NMR 2D spectra were also recorded during NMR titration of ¹⁵N-labelled AVR-Pia with unlabelled OsHPP10-HMA, OsHPP11-HMA or OsHIPP21-HMA (S23 Fig). In these cases, a decrease in intensity was observed for certain peaks, indicating an intermediate exchange regime. This is typical for interactions with weaker affinity (K_D in the micromolar range), and is consistent with the ITC results. Notably, in all three cases, significant changes in intensity were detected for peaks corresponding to residues in the β1, β2 and β3 regions, whereas peaks corresponding to residues on the opposite face remained unaffected. This indicates that similar residues in AVR-Pia are involved in binding to all four HMA domains.

The crystal structure of the AVR-Pia/OsHPP09-HMA complex reveals interface contacts underpinning high affinity binding

To understand the structural detail of the interaction between AVR-Pia and OsHPP09-HMA, we purified and crystallised the effector/HMA complex. Needle-like crystals were obtained in commercial screens and subsequent optimisation of conditions resulted in crystals suitable for diffraction studies. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) to a resolution of 1.65 Å. The structure was solved by molecular replacement, refined and validated as described in the Materials and Methods. Data collection, processing and refinement statistics are presented in S4 Table. The final refined model has been deposited in the Protein Data Bank (PDB accession code 9RSV).

As expected, OsHPP09-HMA adopts the well-characterised HMA domain fold, consisting of a four-stranded antiparallel β-sheet and two ⍺-helices arranged with β⍺ββ⍺β topology (Fig 6). Structures of AVR-Pia have previously been determined by NMR spectroscopy [4,56] (PDB accession codes 2MYW, 2N37) and X-ray crystallography [48] (PDB accession code 6Q76; complex with OsPikp-1 HMA ID). The structure of AVR-Pia determined here is highly similar (RMSD of 0.357 Å to 6Q76, 0.951 Å to 2MYW, and 1.690 Å to 2N37) and adopts the six-stranded β-sandwich fold characteristic of the MAX effectors (Fig 6) [4,6].

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Fig 6. Crystal structure of the HMA domain of OsHPP09 in complex with AVR-Pia.

The structures of OsHPP09-HMA and AVR-Pia are represented as teal and gold ribbons, respectively. The amino acids comprising β2 of AVR-Pia and β2 of the HMA domain are represented as cylinders. Side chains involved in intermolecular hydrogen bonds/salt bridges are also represented as cylinders. Hydrogen bonds are represented by black dashed lines labelled with the bond length. Left insert shows main chain hydrogen bonds between AVR-Pia-β2 and OsHPP09-β2. Right insert shows intermolecular contacts between side chains of residues in OsHPP09-⍺1 and AVR-Pia.

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Globally, the AVR-Pia/OsHPP09-HMA complex resembles the previously determined structures of AVR-Pia/OsPikp-1-HMA (PDB 6Q76) [48] and AVR1-CO39/OsRGA5-HMA (PDB 5ZNG) [12] and the AlphaFold models of AVR-Pia/OsRGA5-HMA and AVR-Pia/OsH(I)PP-HMA complexes (S24 Fig). The AVR-Pia/OsHPP09-HMA interface area is 521.7Å², fractionally larger than AVR-Pia/OsPikp-1-HMA (460.7 Å²) and AVR1-CO39/OsRGA5-HMA (492.8 Å²) (S5 Table). In all the aforementioned complexes, β2 of the MAX effector is aligned with β2 of the HMA domain, forming an antiparallel β-sheet comprising β6, β1, and β2 of the MAX effector and the four-stranded β-sheet of the HMA domain (S24a Fig), with hydrogen bonding between adjacent β-strands (S24b Fig).

In contrast to the crystal structures of AVR-Pia/OsPikp-1-HMA and AVR1-CO39/OsRGA5-HMA and the AlphaFold3 model of AVR-Pia/RGA5-HMA, where the interface is dominated by main chain hydrogen bonding (S24 Fig), in the crystal structure of OsHPP09-HMA/AVR-Pia, the side chains of multiple residues in ⍺1 of the HMA domain form intermolecular hydrogen bonds with the effector. The side chains of OsHPP09^E16 and OsHPP09^K19 form hydrogen bonds with the peptide backbone of AVR-Pia^L38 (S24c Fig). OsPikp-1^R203 is in the corresponding position to OsHPP09^K19, however it is OsPikp-1^R226, in the β2-β3 loop, which forms an analogous hydrogen bond with AVR-Pia^L38 (S24c Fig). OsRGA5-HMA has a basic residue (R1012) at the corresponding position to OsHPP09^K19, and the side chain of OsRGA5-HMA^R1012 is orientated towards the AVR1-CO39^D35 side chain, however the distance between them (4.1 Å) is greater than the 4 Å cut-off typically used when considering salt bridge interactions (S24c Fig). At the start of β2, the OsHPP09^D33 side chain forms salt bridge interactions with the AVR-Pia^R43 side chain (S24d Fig). This aspartate residue is conserved in both OsPikp-1-HMA (D217), where it similarly forms a salt bridge with AVR-Pia^R43, and OsRGA5-HMA (D1026), where it forms an indirect, water-mediated contact with the backbone of AVR1-CO39^T41 (S24d Fig). The OsHPP09^Q20 side chain forms hydrogen bonds with the side chain hydroxyl of AVR-Pia^Y41 (S24e Fig). The serine in the corresponding position in OsPikp-1-HMA (S204) forms a water-mediated contact with the side chain hydroxyl of AVR-Pia^Y41 (S24e Fig). Finally, towards the C-terminal end of OsHPP09-⍺1, the side chain of OsHPP09^D28 forms a salt bridge with AVR-Pia^R23 (S24f Fig). By contrast, the corresponding amino acid in OsPikp-1, OsPikp-1^S212, forms a hydrogen bond with the side chain hydroxyl of AVR-Pia^Y85 (S24f Fig). Overall, the increased number and altered arrangement of intermolecular hydrogen bonds in the AVR-Pia/OsHPP09-HMA structure compared to the structure of AVR1-CO39/OsRGA5-HMA likely explains the difference in binding affinities.

Comparisons revealed that the crystal structure of OsHPP09-HMA/AVR-Pia was highly similar to the AlphaFold2 and AlphaFold3 models (global RMSD of 0.514 Å and 0.606 Å, respectively). Of the 12 hydrogen bonds and salt bridges (determined by qtPISA [57], 2.5Å-3.5 Å) in the experimental structure, 8 were present in the AlphaFold2 model and 9 in the AlphaFold3 model, with differences largely due to variation in the orientation of side chains (S25 and S26 Figs). For example, the AVR-Pia^R23 sidechain is predicted to form a hydrogen bond with the sidechain of OsHPP09-HMA^D28 by AlphaFold3, and with the backbone of OsHPP09-HMA^A27 by AlphaFold2. In the crystal structure, the positions of the AVR-Pia^R23 and OsHPP09-HMA^D28 sidechains enables AVR-Pia^R23 to form hydrogen bonds with both OsHPP09-HMA^D28 and OsHPP09-HMA^A27 (S25 and S26 Figs). As AlphaFold does not predict the positions of water molecules, the AlphaFold models lack the water-mediated interface contacts at the interface which can be identified in the crystal structure.

Replacement of OsHPP09^E16 with alanine or arginine disrupts AVR-Pia binding

We aimed to identify point mutations in OsHPP09-HMA that interfere with AVR-Pia binding. First, we selected residues whose side chains contribute intermolecular hydrogen bonds or salt bridges and mutated these to alanine (E16A, K19A, Q20A, D28A, D33A) and, for acidic residues, to arginine (E16R, D28R, D33R). D33 is conserved in OsHPP09, OsHPP10, OsHPP11, and in the HMA IDs of OsPikp-1 (D217) and OsRGA5 (D1026) (S5 and S24d Figs). By contrast, the corresponding residue in OsPikm-1-HMA, which does not bind AVR-Pia [48], is histidine. We therefore also mutated D33 to histidine. Second, we used FoldX [58] to predict the effect of mutating each interface residue in OsHPP09-HMA to all other amino acids. Based on the calculated difference of interaction free energy (ΔΔG) upon mutation, we made three further mutations: K19W, I35K, and A37W.

Introduction of the point mutations E16A, E16R, D28A, D28R and I35K in OsHPP09 resulted in a complete loss of interaction with AVR-Pia in Y2H experiments (Fig 7a). The remaining mutations (i.e., K19A, K19W, D33A, D33H, D33R, A37W) with the exception of Q20A, led to reduced yeast growth on selective media suggesting a weakening of the interaction compared to the wildtype OsHPP09/AVR-Pia pair (Fig 7a). Immunoblot analysis revealed that the K19A, K19W and I35K mutants exhibited lower protein expression levels, while all other mutant constructs showed similar expression to wildtype OsHPP09 (S4d Fig).

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Fig 7. Mutation of OsHPP09 E16 reduces AVR-Pia binding.

a Y2H interaction analysis between AVR-Pia (∆SP) and full-length (FL) OsHPP09 carrying single point mutations. OsHPP09^WT/AVR-Pia served as positive control. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH and SD/-LWH supplemented with 5 mM 3AT). Photos were taken after 7 days of incubation. AD, activating domain; BD, binding domain. b Split luciferase complementation assays of AVR-Pia (∆SP; fused to the N-terminal part of luciferase; 3xHA:NLuc:AVR-Pia) with FL OsHPP09 carrying indicated mutations (fused to the C-terminal part of luciferase: 3xFlag:CLuc:OsHPP09). Co-expression of AVR-Pia with OsHPP09 served as a positive control, co-expression of OsHIPP43 with AVR-Pia as a negative control. Leaves infiltrated with P19 alone served as background (BG) control. Box plots show the median (line), mean (cross) and upper/lower quartiles (box limits), whiskers extend to the most extreme data points within 1.5x the interquartile rang, with outliers plotted individually. Three independent replicates were performed, with n = 8 per combination per replicate. Data points are shown with shapes corresponding to the replicate. Different letters indicate statistically significant differences based on a pairwise Wilcoxon test (α = 0.05). c Circular dichroism (CD) spectra obtained for WT OsHPP09-HMA (pale blue) and variants carrying E16A, E16R, I35K or D33H mutations. Negative peaks at 222 nm/208 nm and a positive peak at 193 nm indicate ⍺-helices while a negative peak at 218 nm and positive peak at 195 nm indicate β-sheets, consistent with the β⍺ββ⍺β HMA domain topology. Low ellipticity above 210 nm and a negative peak at 200 nm indicates disorder. d Overlays of cross-peaks from HSQC spectra corresponding to residues G30, I44, E56 and Y85 of ¹⁵N-labelled AVR-Pia alone (orange) and in the presence of either WT OsHPP09-HMA or variants carrying E16A, E16R or D33H mutations (blue/green/purple). Overlays of the full spectra are provided in S29–S31 Figs.

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To assess the effect of the mutations in the context of the full-length protein in planta, we performed split-luciferase complementation assays. The E16A and E16R mutants showed significantly reduced interaction with AVR-Pia, while the I35K mutant completely lost its ability to interact with AVR-Pia, consistent with the Y2H results (Fig 7b). In contrast, the D28A and D28R mutations, which abolished interaction in Y2H assays, had only a minor effect on the interaction with AVR-Pia in planta, similar to the other tested mutants (Fig 7b). Immunoblot analysis demonstrated that all proteins were expressed at comparable levels in planta (S27 Fig).

Disruption of AVR-Pia binding to the mutated HMA domains may be caused by a specific loss of interaction at the interface or by broader destabilisation of the HMA domain fold. We therefore aimed to establish whether the E16A, E16R, D33H and I35K mutations affect the structure of the HMA domain. Mutated HMA domains were produced in E. coli and purified to homogeneity (S28 Fig), and molecular weights were confirmed by intact mass spectrometry. Circular dichroism (CD) spectra for OsHPP09^E16A-HMA, OsHPP09^E16R-HMA and OsHPP09^D33H-HMA were comparable to the spectra for wildtype OsHPP09-HMA with features characteristic of ⍺-helices (negative peaks around 222 nm and 208 nm, positive peak around 193 nm) and β-sheets (negative peak around 218 nm, positive peak around 195 nm) consistent with the HMA domain fold (Fig 7c). By contrast, OsHPP09^I35K-HMA showed features of disorder (low ellipticity above 210 nm and a negative peak around 200 nm) (Fig 7c), suggesting that the lack of AVR-Pia binding to the I35K mutant is likely due to disruption of the HMA domain fold.

We obtained ¹H-¹⁵N-HSQC NMR spectra of ¹⁵N-AVR-Pia in the presence of (unlabelled) HMA domains of OsHPP09^E16A, OsHPP09^E16R, and OsHPP09^D33H. The spectra of ¹⁵N-AVR-Pia with OsHPP09^D33H-HMA was highly similar to the spectra of ¹⁵N-labelled AVR-Pia with WT OsHPP09-HMA (Figs 7d and S29), although the exchange regime changes at certain cross-peaks, suggesting lower binding affinity. By contrast, the spectra of ¹⁵N-AVR-Pia with OsHPP09^E16A or OsHPP09^E16R showed no chemical shift perturbation or intensity diminution and largely superimposed with the spectra of ¹⁵N-AVR-Pia alone (Figs 7d and S30 and S31). Taken together, these results demonstrate that E16A and E16R mutations block AVR-Pia binding without disrupting the HMA fold, while the I35K mutation interferes with correct folding of the HMA domain.

Given the overlap between the AVR-Pia-binding surface of OsHPP09-HMA and the putative dimerization interface, we used analytical size exclusion chromatography to investigate whether OsHPP09^E16A or OsHPP09^E16R dimerise in solution. Both mutated HMA domains eluted at a similar volume to WT OsHPP09-HMA, suggesting that the mutation does not interfere with dimerization (S32 Fig).

Residues corresponding to OsHPP09^E16 in OsHPP10 and OsHIPP21 are important for AVR-Pia binding

Despite differing by an order of magnitude in their affinity for AVR-Pia binding, OsHPP10-HMA and OsHPP09-HMA vary in just six amino acid positions. Intriguingly, the only residue of the six that is located at the interface with AVR-Pia is OsHPP09^E16/OsHPP10^D16 (S5 Fig). To test whether this polymorphism is responsible for the observed difference in strength of binding to AVR-Pia, we generated the reciprocal mutants OsHPP09^E16D and OsHPP10^D16E and tested for interaction with AVR-Pia in pairwise Y2H assays. Strong yeast growth on interaction-selective media was observed when AVR-Pia was paired with WT OsHPP09 (HMA or FL), or OsHPP10^D16E (HMA or FL). By contrast, no yeast growth was observed for OsHPP09^E16D nor OsHPP10 (S33a Fig). Expression of all proteins was confirmed by immunoblot analyses (S33b Fig).

To establish the importance of the residue corresponding to OsHPP09^E16 in OsHPP10 (D16), OsHPP11 (T16) and OsHIPP21 (E15) for the interaction of the H(I)PPs with AVR-Pia, we mutated each residue to alanine (A) and arginine (R) and tested for an interaction with AVR-Pia in planta using split luciferase complementation assays. Consistent with the effect observed for the E16A and E16R substitutions in OsHPP09, mutation of the equivalent residue in OsHPP10 and OsHIPP21 significantly reduced their interaction with AVR-Pia. In contrast, the T16A and T16R substitutions in OsHPP11 did not significantly impact AVR-Pia binding (S34a Fig). Immunoblot analysis confirmed that all proteins were expressed at detectable levels (S34b Fig).

Discussion

In the present study, we found that the M. oryzae MAX effector AVR-Pia binds to the HMA domains of four rice H(I)PPs: OsHPP09, OsHPP10, OsHPP11, and OsHIPP21. Neither AVR-Pik, Pwl2 nor AVR1-CO39 interact with these H(I)PPs, demonstrating that AVR-Pia binds a distinct group of H(I)PPs to those previously identified as MAX effector targets. AVR-Pik interacts with multiple H(I)PPs within a large phylogenetic clade (referred to as “clade A”) [27], while Pwl2 interacts specifically with OsHIPP43 and its homologs from other grass species [9].

Like AVR-Pia, AVR1-CO39 is recognised by direct binding to the HMA ID of OsRGA5. Although the interaction surfaces between AVR-Pia/OsHPP09-HMA and AVR1-CO39/OsRGA5-HMA share similarities (S24 Fig), as the β2 strand of the MAX effector interacts with the β2 strand of the HMA domain, the crystal structure of AVR-Pia/OsHPP09-HMA reveals that the side chains of several residues in the ⍺1 helix of the HMA domain form intermolecular hydrogen bonds with the effector. In contrast, the AVR1-CO39/OsRGA5-HMA interface is predominantly mediated by hydrogen bonding between the main chains. Interestingly, AVR1-CO39 did not interact with the same OsH(I)PPs as AVR-Pia. AVR1-CO39 also did not interact with the “clade A” OsH(I)PPs bound by AVR-PikD [27]. AVR1-CO39 is broadly absent from rice-infecting isolates of M. oryzae [7,59,60] and its loss has potentially contributed to a host shift from foxtail millet (Setaria sp.) to rice. It is therefore possible that AVR1-CO39 has evolved to target H(I)PPs from other grass species, and does not interact with those present in rice.

Strikingly, the interactions between AVR-Pik, Pwl2, and AVR-Pia and HMA domains of the H(I)PPs bound by these effectors involve distinct interfaces. In the crystal structures of AVR-Pia/OsHPP09-HMA and Pwl2/OsHIPP43-HMA, β2 of the core MAX fold and β2 of the HMA domain are aligned in an antiparallel orientation [9], forming an antiparallel β-sheet comprising the four β-strands of the HMA domain and β2, β1, and β6 of the MAX fold. When the HMA domains are superposed, the relative positions of β2 of the MAX fold of Pwl2 and AVR-Pia are very similar (S35 Fig). However, the other β-strands of Pwl2 are shifted compared to AVR-Pia, enabling residues in β3, β4, and the β3-β4 loop of the MAX fold to contribute to the HMA-binding interface. By contrast, in the OsHIPP19-HMA/AVR-PikF complex, an antiparallel β-sheet forms from the four β-strands of the HMA domain and β3, β4 and β5 of the MAX fold, mediated by hydrogen bonding between β3 of AVR-PikF and β4 of HIPP19-HMA [8]. Furthermore, while AVR-Pia consists solely of the core MAX fold, AVR-Pik has an unstructured extension at the N-terminus, while in Pwl2 the characteristic MAX fold is followed by an ⍺-helix and a C-terminal unstructured region, and these extensions are involved in HMA binding (S35 Fig). Pwl2^E89, in the ⍺-helix, forms a hydrogen bond with OsHIPP43-HMA^K51, while the C-terminal unstructured segment of Pwl2 extends across the HMA domain, forming an extensive interface involving both hydrogen bonds and π-stacking interactions. Similarly, the N-terminal extension of AVR-Pik forms multiple hydrogen bonds with residues in β3, β2, and the β2-β3 loop of OsHIPP19-HMA. The interface in the AVR-Pia/OsHPP09-HMA complex is smaller than in AVR-PikF/OsHIPP19-HMA and Pwl2/OsHIPP43-HMA (521.6 Å² compared to 1044.3 Å² and 1965.2 Å², respectively), and is formed almost exclusively by residues in β2 of the core MAX fold. It is highly interesting that at least three MAX effectors have evolved to bind HMA domains in different ways. This highlights how structural variation within this effector family can underpin different interactions with host targets. It is unknown whether the interactions of AVR-Pia, AVR-Pik, and Pwl2 with OsH(I)PPs lead to similar outcomes (redundancy) or if each binding event has a distinct and specific impact on plant susceptibility and/or fungal virulence.

While AVR-Pia, AVR-Pik, and Pwl2 all target OsH(I)PPs, the MAX effector AvrPiz-t has been reported to interact with a range of host proteins [61,62]. From a structural perspective, it is unclear how a single MAX effector binds to such diverse targets. However, this suggests that MAX effectors may target many different host proteins and processes during infection.

AVR-Pia is recognised by the paired rice NLR immune receptors OsRGA5 and OsRGA4 through direct binding of the effector to the non-canonical HMA domain integrated into OsRGA5 [11]. Such non-canonical domains are hypothesised to have originated in effector host targets and were incorporated into NLR proteins to function as effector “sensors” [15]. Consistent with this, the crystal structure of AVR-Pia/OsHPP09 revealed that AVR-Pia binds OsHPP09-HMA and OsRGA5-HMA through similar interfaces [13]. Interestingly, the F24S and T46N polymorphisms of AVR-Pia-H3 interfered with binding to both OsRGA5-HMA and the candidate OsH(I)PP targets, in contrast to naturally-occurring polymorphisms in AVR-Pik which prevent binding to the HMA ID of OsPikp-1 without disrupting interaction with the HMA domains of OsH(I)PP19 and OsH(I)PP20 [8,27]. Both AVR-Pia^F24 and AVR-Pia^T46 are located at the HMA binding interface of AVR-Pia. AVR-Pia^F24 contributes to a hydrophobic patch on AVR-Pia, and the replacement of phenylalanine by a polar serine residue presumably disrupts this hydrophobic patch. This substitution alone was sufficient to disrupt the interaction between AVR-Pia and OsRGA5-HMA [13]. Exchange of AVR-Pia^T46 for the slightly larger asparagine may introduce steric hindrance, increasing the distance between the effector and HMA domain.

Interestingly, phylogenetic analysis of rice H(I)PP proteins (including NLRs with an HMA ID), based on the alignment of their HMA domains clusters OsRGA5-HMA within Clade A, which notably includes the H(I)PPs targeted by AVR-Pik and the HMA ID of OsPik-1 [27]. In contrast, the HMA domains of OsHPP09, OsHPP10, OsHPP11, and OsHIPP21 do not belong to this clade and are phylogenetically distant from that of OsRGA5. This indicates that the HMA ID of OsRGA5 did not arise through duplication and integration of these AVR-Pia candidate targets, but instead originated from another H(I)PP, likely belonging to Clade A. Future work aimed at reconstructing the evolutionary history of OsRGA5 will be necessary to further address this question. However, the fact that OsRGA5-HMA is able to bind AVR-Pia via an interface similar to that used by AVR-Pia to bind OsHPP09 (S24 Fig) suggests a case of convergent evolution of the interface structures.

A previous study reported that AVR-Pia binds OsRGA5-HMA with a K_D of 7.8 μM [13]. This is comparable to the binding affinity of AVR-Pia to OsHPP10-HMA, but at least one order of magnitude weaker than the AVR-Pia/OsHPP09-HMA interaction. This higher affinity can be explained by the number of intermolecular hydrogen bonds and salt bridges at the AVR-Pia/OsHPP09 interface; available structures of AVR1-CO39/OsRGA5-HMA and AVR-Pia/OsPikp-1-HMA, combined with modelling approaches, suggest that the AVR-Pia/OsRGA5-HMA interface likely involves fewer intermolecular contacts. A similar pattern was reported for AVR-Pik/HMA interactions; surface plasmon resonance experiments showed that AVR-PikD interacts with the HMA domain of OsHIPP19 with higher affinity than the HMA ID of OsPik-1 [8]. It is important to note that these binding affinities are determined in vitro using purified HMA domains. These experiments therefore lack the biological context of the FL protein or the plant cell environment.

We observed a discrepancy in OsHPP10-AVR-Pia interaction between Y2H and split-luciferase assays, with interaction detected in split-luciferase but not in Y2H. This likely reflects differences in assay sensitivity and stringency rather than a true absence of interaction [63]. Y2H requires stable interaction and transcriptional activation in the yeast nucleus and is generally more suited to detecting relatively strong interactions. In contrast, split-luciferase complementation is a highly sensitive in planta assay that benefits from proximity-induced signal amplification and can detect lower-affinity interactions. ITC showed that AVR-Pia binds OsHPP09-HMA with high affinity (K_D = 115 nM), whereas binding to OsHPP10-HMA is substantially weaker (K_D = 5.26 µM), with OsHPP11-HMA and OsHIPP21-HMA showing intermediate affinities. This affinity series is consistent with assay-dependent detection patterns. Stronger interactions (AVR-Pia with OsHPP09, OsHPP11, and OsHIPP21) are detected in Y2H, whereas weaker micromolar interactions fall below the detection range of Y2H but are detected in split-luciferase due to its higher sensitivity.

In recent years, advances in structural modelling have offered new insights and driven new lines of research in molecular plant-microbe interactions. Given reports that AlphaFold confidence metrics can be used to discriminate interacting protein pairs from those which do not interact [50,51,64], we were curious as to whether AlphaFold could identify the subset of H(I)PPs bound by AVR-Pia. Almost all AVR-Pia/HMA complexes, real and imagined, were modelled with high confidence scores to bind at a similar interface. The presence of an AVR-Pia/HMA structure in the PDB, used in the training of AlphaFold2 and AlphaFold3, may have influenced the results, highlighting a potential limitation of AlphaFold modelling. However, the intermolecular contacts were broadly well predicted, with a few discrepancies resulting from differences in the orientation of side chains. It should also be noted that the crystal structure of a protein (or protein complex) reflects a conformation of the protein that is amenable to crystallisation, which may not be identical to the structure of the protein in solution.

H(I)PPs, which form extensive families in plants [19,25,26], have been reported to be involved in intracellular metal transport and homeostasis, responses to cold and drought stress, and heavy metal tolerance. In addition to the interactions between M. oryzae MAX effectors and OsH(I)PPs, there are multiple examples of plant H(I)PPs targeted by proteins from diverse pests and pathogens. For example, the putative effector RsMf8HN from the necrotrophic fungus Rhizoctonia solani interacts with OsHIPP28 [38], while a HIPP from sugarcane (ScPi21) is a target for the Fusarium sacchari effector Fs00367 [35]. CsHIPP03 was identified by Y2H screening as an interactor of SDE34, an effector secreted by the phloem-colonising bacteria Candidatus Liberibacter asiaticus [40]. A root-knot nematode (Meloidogyne graminicola) effector binds rice OsHPP04 and suppresses the flg22-induced ROS burst [37], while the potato mop-top virus movement protein interacts with NbHIPP26 to promote systemic spread of the virus [31]. Further, the non-MAX M. oryzae effector AVR-Pita interacts with a HIPP HMA domain designated OsHMA120 [65]. Replacing the HMA-ID of OsRGA5 with OsHMA120 resulted in an engineered NLR protein conferring resistance to AVR-Pita. Together, this indicates that the manipulation of H(I)PPs is required for infection by diverse pests and pathogens and that H(I)PPs are targeted by structurally diverse pathogen proteins. Understanding how pathogen effectors bind H(I)PPs can offer opportunities to engineer novel resistance phenotypes [66]. However, little is known about the molecular mechanisms through which H(I)PPs affect plant resistance and/or susceptibility, and the precise functions of the effectors which target H(I)PPs.

HMA domains typically coordinate metal ions via two cysteine residues in an MxCxxC motif. Intriguingly, the metal-binding motif is degenerate in all four HMA domains bound by AVR-Pia, with both cysteines replaced by other amino acids (MTDEKT in OsHPP09, MTDDKI in OsHPP10, MSDTKM in OsHPP11, and MTDERK in OsHIPP21), indicating the absence of metal-binding. Consistent with this, no electron density suggestive of bound metal ions was observed in the OsHPP09-HMA/AVR-Pia crystal structure. Interestingly, OsHPP09^E16, which is important for AVR-Pia binding, is located in the degenerate metal binding motif. Of the “clade A” H(I)PPs bound by AVR-Pik, some contain an intact MxCxxC motif, while others, such as OsHIPP19 and OsHIPP20, lack one or both cysteine residues. OsHIPP43 has an intact MxCxxC motif, though it is unknown whether it binds metal ions in planta. In cases where HMA domains lack a complete MxCxxC motif, and presumably also the capacity to bind metals, the functions of the H(I)PPs are particularly intriguing. Functional diversification, beyond metal transport and homeostasis, may have been facilitated by the stable core fold of the HMA domain supporting diversification of surface properties to deliver new functions. Protein prenylation can affect subcellular localisation, protein stability, and protein-protein interactions [67]. For some HIPPs, the C-terminal CaaX isoprenylation motif has been shown to drive localisation to plasmodesmata [27,34]. Further investigation is required to establish the localisation and function of the AVR-Pia interactors in planta.

Pathogens require certain host factors to establish a successful infection, with the underlying genes typically referred to as susceptibility (S) genes [68]. Previous studies have shown that OsHIPP05 (Pi21) and OsHIPP20 are S genes for blast infection; oshipp05 and oshipp20 CRISPR-Cas9 knockout plants exhibited increased blast resistance compared to WT plants [27,33,69–71]. Further, a recent study reported that overexpression of barley (Hordeum vulgare) HvHIPP43 increased susceptibility to M. oryzae, suggesting that HvHIPP43 may also be an S gene [34]. Other plant H(I)PPs are also S genes for different pests and pathogens [30,32,36]. It remains to be determined whether OsHPP09, OsHPP10, OsHPP11, and/or OsHIPP21 are also S genes for rice blast disease, and whether deletion of any of these genes increases blast resistance. Notably, AVR-Pia contributes to virulence on rice [10]. Targeted mutagenesis of OsH(I)PPs to prevent AVR-Pia binding may reduce pathogen virulence without compromising the endogenous function of OsH(I)PPs. However, the extent of functional specificity and redundancy of both OsH(I)PPs and the effectors targeting them remains unknown, and may hamper efforts to improve resistance through modification of OsH(I)PPs.

Structure-guided mutagenesis of OsHPP09 identified that a single point mutation, E16A or E16R, could prevent AVR-Pia binding without perturbing the HMA domain fold. The OsHPP09^E16 side chain forms an intermolecular hydrogen bond with the backbone of AVR-Pia and a salt bridge with the side chain of OsHPP09^K19, which also forms a hydrogen bond with the AVR-Pia backbone. This arrangement of hydrogen bonds likely stabilises the interaction between the two proteins and contributes to the high affinity binding of OsHPP09-HMA by AVR-Pia. By contrast, in the AlphaFold model of OsHPP10-HMA/AVR-Pia, the shorter aspartate side chain is too far from the AVR-Pia backbone to form an intermolecular hydrogen bond (S36 Fig). This polymorphism is sufficient to significantly reduce the affinity of OsHPP10-HMA for AVR-Pia. However, mutating the aspartate to either alanine or arginine appears to further reduce the strength of interaction between OsHPP10 and AVR-Pia. The residue corresponding to OsHPP09^E16 in OsHPP11 is threonine (T16); in the AlphaFold model of OsHPP11-HMA/AVR-Pia, the corresponding threonine side chain is too far from the effector to form an intermolecular hydrogen bond. As the side chains of both aspartate and threonine are hydrogen bond acceptors, it is possible that a water molecule could bridge the space between these residues and the AVR-Pia backbone. However, mutating the threonine to either alanine or arginine did not significantly affect the interaction between OsHPP11 and AVR-Pia. The glutamate residue is conserved in the HMA domain of OsHIPP21 (E15) and likely supports a similar network of hydrogen bonding to that observed in the OsHPP09-HMA/AVR-Pia crystal structure. This may explain why, despite lacking the D33 (L32 in OsHIPP21), Q20 (S19 in OsHIPP21) and D28 (K27 in OsHIPP21) residues which are conserved in the three HPPs and each form intermolecular hydrogen bonds in the OsHPP09-HMA/AVR-Pia structure, OsHIPP21-HMA is still bound by AVR-Pia. Consistent with this, mutation of OsHIPP21^E15 to alanine or arginine reduced the strength of the interaction with AVR-Pia.

Despite the predicted overlap of the dimerization interface with the effector binding surface, the E16A and E16R mutants do not appear to disrupt dimerization of OsHPP09-HMA. The AlphaFold3 model of the OsHPP09-HMA dimer predicts that the side chain of K19 forms an intermolecular salt bridge with the side chain of D33 of the opposing protomer. E16 is not directly involved in intermolecular bonds at the dimerization interface, though the side chain of E16 forms an intramolecular salt bridge with the side chain of neighbouring K19 which may stabilise the interface, and could also be involved in water-mediated contacts between protomers which are not revealed by AlphaFold modelling. While dimerization of HMA-IDs is not required for OsPik-1- or OsRGA5-mediated effector recognition and immune activation, it is unknown whether dimerization of OsHPP09-HMA holds biological significance. If dimerization is related to function, mutations that block effector binding without disrupting dimer formation could be of interest for engineering.

In summary, we have characterised the interactions between AVR-Pia and a novel subset of HMA domain-containing rice proteins. Biophysical and structural analyses guided mutagenesis of these effector targets and identified a mutation that blocks effector binding without compromising structural integrity of the HMA domain. This provides a foundation for targeted modification of these proteins for enhanced disease resistance.

Methods

Cloning

The coding sequences (CDS) of OsH(I)PPs, including the stop codon, were synthesised by Twist Bioscience. The CDS of OsHIPP14, OsPi21, OsHIPP19, OsHIPP21, and OsHPP09 were codon-optimised to enable synthesis.

For Y2H and split luciferase experiments, synthesised OsH(I)PP genes were used as template for amplification with primers containing attB1 and attB2 recombination sites (S6 Table) and Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Resulting PCR products were subsequently introduced into the pDONR207 vector (Invitrogen) via Gateway BP recombination (Thermo Fisher Scientific). Entry constructs for the MAX effectors dSP-AVR-Pia [15], dSP-AVR1-CO39 [12], dSP-AVR-PikD [47] and dSP-Pwl2 [13] were previously published. The dSP-AVR-PikC fragment was amplified from pCambia1300_dSP-AVR-PikC [45] and dSP-MAX58 from a synthesised gene, both using primers containing attB1 and attB2 recombination sites (S6 Table) and Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Resulting PCR products were cloned into the pDONR207 vector (Invitrogen) via Gateway BP recombination (Thermo Fisher Scientific).

For recombinant protein production in E. coli, synthesised genes were used as templates for PCR amplification of HMA domains (S1 Table) with primers containing BpiI restriction sites (S6 Table) using CloneAmp HiFi PCR Premix (Clontech Laboratories). Resulting PCR products were introduced into pICH41308 (Addgene #47998 [72]) by Golden Gate assembly [73] to generate level 0 CDS modules. The CDS of OsHIPP21-HMA was codon-optimised for E. coli expression and supplied with appropriate overhangs and restriction sites for direct assembly into the vector pICH41308 without PCR amplification. Level 0 CDS modules were used in Golden Gate reactions with the pOPIN-GG vector pPGN-C (Addgene #174578 [74]) and the level 0 module pICSL30015 (Addgene #174582 [74]) to generate E. coli expression constructs with a cleavable N-terminal 6xHis-MBP tag.

Site-directed mutagenesis of OsHPP09 FL for Y2H and split luciferase, and OsHPP09-HMA for recombinant protein production, as well as for OsHPP10, OsHPP11, OsHIPP21 was performed using a QuikChange Site-Directed Mutagenesis Kit (Agilent). Prenylation motif mutations (OsHIPP14^C187S, OsHIPP21^C133S, OsHIPP39^C190S, OsHIPP41^C152S) in the pDONR constructs, and mutations in AVR-Pia to give AVR-Pia-H3, were introduced by site-directed mutagenesis PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and mutation-specific primers listed in S6 Table.

Gateway LR recombination using LR Clonase II mix (Thermo Fisher Scientific) was used to transfer OsH(I)PPs and OsRGA5_Cter [11] (883–1116 aa) into Gateway-compatible destination vectors, including a modified Y2H vector pGBKT7-GW [75] and the plant expression vector pBIN-35S-3Flag-Cluc-GWY (provided by L. Deslandes). Similarly, MAX effector pDONR constructs were recombined into a modified pGADT7-GW [75] and pBIN-35S-3HA-Nluc-GWY (provided by L. Deslandes) destination vectors. The pBIN-35S-3HA-Nluc-GWY and pBIN-35S-3Flag-Cluc-GWY vectors were generated by ligation of a 3HA-Nluc-FrameB or 3Flag-Cluc-FrameB fragment, respectively, into a XhoI/XbaI digested pBIN-35S vector.

Previously published expression constructs for AVR1-CO39, AVR-Pia, and AVR-Pia-H3 [4,12,13] were used for protein production in the present study.

A complete list of plasmids used in this study is provided in S7 Table.

Yeast two-hybrid

The yeast strain Y2HGold (mating type a; Takara Bio) was transformed with the bait construct expressed from the pGBKT7 vector, which contains the GAL4-BD and the TRP1 marker gene. These constructs included previously described HMA domains [49], as well as newly generated constructs: OsHIPP21-HMA, OsH(I)PP^WT FL,OsH(I)PP^C/S FL, OsHPP09^E16D and OsHPP10^D16E. The yeast strain Y187 (mating type α; Takara Bio) was transformed with the prey construct (MAX effectors) expressed from the pGADT7 vector, which carries the GAL4-AD and the LEU2 marker gene. Yeast transformation was performed using a lithium acetate (LiAc)-based protocol according to the Yeastmaker Yeast Transformation System (Takara Bio). Transformed yeast cells were plated on synthetic defined (SD) media lacking either tryptophan (-W) or leucine (-L) and incubated at 30 °C for 3 days.

Mating of the transformed yeast strains followed the Matchmaker Gold Yeast Two-Hybrid System User Manual (Takara Bio). Diploid yeast cells were selected on SD/-L-W plates (SD medium lacking leucine and tryptophan) after incubation at 30 °C for 2 days. For Y2H assays, diploid yeast cells were inoculated in SD/-L-W medium, grown for 24 hours at 28°C with shaking at 100 rpm, then pelleted and washed twice with sterile water. Serial 10-fold dilutions (10^‒1, 10^‒2, and 10^‒3) were spotted onto SD selection plates: SD/-L-W (growth control), SD/-L-W-H (lacking leucine, tryptophan, and histidine), SD/-L-W-H supplemented with varying concentrations of 3-amino-1,2,4-triazole (3AT). After 7 days incubation at 30 °C, plates were photographed using the presentation station UF-130DX (Samsung).

Yeast two-hybrid cDNA library screening

The Y2H cDNA library derived from the rice cultivar CO39 and the BD:dSP-AVR-Pia construct, have been previously described [11,47]. Library screening followed the Matchmaker Gold Y2H system protocol (Takara Bio). Mating was performed between the Y2H Gold strain harbouring GAL4-BD:AVR-Pia and the Y187 strain carrying the CO39 cDNA library. Diploid yeasts were plated on SD/-L-W-H medium and supplemented with 20 mM 3AT. Colonies that grew were re-streaked onto fresh SD/-L-W-H medium containing 20 mM 3AT to confirm interaction-dependent growth. Yeast colony PCR was used to amplify cDNA inserts from positive clones, and PCR products were sequenced. Sequences were aligned to the cDNAs of two publicly available annotations of the Oryza sativa cv. Nipponbare reference genome (IRGSP-1.0 [76] and MSU7.0 [77]) using BLAST to identify the corresponding genes. Only genes for which the cDNA inserts (or fragments of cDNAs) were cloned in-frame with the GAL4-AD were retained as candidate AVR-Pia targets.

Agrobacterium-mediated transient expression in N. benthamiana

Agrobacterium tumefaciens strains AGL1 (carrying split-luciferase constructs) and GV3101-pMP90 (carrying 35S::P19) were grown overnight at 28°C in 5 mL LB medium supplemented with appropriate antibiotics. Overnight cultures were pelleted at 4,000 x g for 10 minutes, and pellets resuspended in 2 mL infiltration buffer (10 mM MgCl₂, 10 mM MES pH 5.6, 150 µM acetosyringone). Bacterial suspensions were incubated in the dark for 2 hours. The optical density at 600 nm (OD₆₀₀) was adjusted to 0.05 for 35S::P19 and 0.1 for OsH(I)PPs and MAX effectors, respectively. In all infiltration mixes, 35S::P19 was co-infiltrated to suppress gene silencing. Agrobacterial suspensions were mixed and infiltrated into leaves of 5–6-week-old N. benthamiana plants using a needleless syringe. Leaf discs were harvested 2 days post-infiltration for luminescence measurements or protein extraction. N. benthamiana plants were grown in a growth chamber on soil under a 16-hour light/ 8-hour dark cycle, at 20 °C and 65–75% relative humidity.

Split luciferase assay

For luminescence measurements, 4 mm leaf discs (collected using a Miltex Biopsy Punch (Delta Microscopie)) were placed into wells of a white 96-well plate containing water. The water was removed and replaced with a 1 mM Xenolight D-luciferin-K⁺ salt bioluminescent substrate solution (Perkin Elmer). Luminescence was quantified using a Spark microplate reader (Tecan) over three cycles of 10 minutes each. For all experiments, data from the third cycle were used for analysis. Each interaction was tested with six to eight technical replicates (six or eight leaf discs per plate from four individual plants per biological replicate, unless otherwise indicated in the figure legend). The luminescence assay was performed in three independent experiments. Statistical analyses from three independent replicates (24 leaf discs in total, unless otherwise indicated in the figure legend) and data visualisation were done using R (version 4.4.1). Multiple comparisons were performed using pairwise Wilcoxon tests followed by a Benjamini-Hochberg p-value adjustment with the stats package. Boxplots were generated using the ggplot2 package.

Protein extraction and Western blotting

Protein extraction from yeast was performed using a post-alkaline extraction protocol, as described by Kushnirov [78]. For BD-OsHIPP14/19/20/39/41 and AD-MAX, yeast crude extracts were resuspended in 50 µL of 1x LDS loading buffer (1x Bolt LDS sample buffer (Thermo Fisher Scientific), 1x NuPAGE sample reducing agent (Thermo Fisher Scientific)), boiled at 95°C for 10 minutes, and loaded onto a 10% NuPAGE Bis-Tris gels (Invitrogen). For BD-OsHPP09/10/11 and OsHIPP21, yeast crude extracts were resuspended in 50 µL of 1x SDS-Tricine buffer (1x SDS-Tricine buffer (Thermo Fisher Scientific), 1x NuPAGE sample reducing agent (Thermo Fisher Scientific)) and boiled at 85 °C for 2 minutes before loading onto a 10–20% Tricine gel (Novax). Proteins were transferred to iBlot nitrocellulose membrane (Invitrogen) using the iBlot 2 Gel Transfer Device (program: 20 V 1 min, 23 V 4 min, 25 V, 2 min, Thermo Fisher Scientific), and analysed by immunoblotting.

For protein extraction from N. benthamiana, four leaf discs (0.8 mm diameter) were collected, immediately frozen in liquid nitrogen and ground using a tissue homogeniser (Qiagen). The ground plant material was solubilised in 200 µL extraction buffer (20 mM Tris-Hcl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, 5 mM DTT, 1x Protease inhibitor cocktail (Sigma-Aldrich), 1x EDTA-free cOmplete protease inhibitor (Roche)). Protein extracts were centrifuged at 18,000 x g for 15 minutes at 4 °C, and the resulting supernatants retained. 2x LDS buffer (Bolt LDS sample buffer (Thermo Fisher Scientific) and NuPAGE sample reducing agent (Thermo Fisher Scientific)) was added and samples were denatured at 70 °C for 10 minutes. Proteins were resolved by electrophoresis on NuPAGE 4–12% Bis-Tris gels (Invitrogen). Proteins were transferred to iBlot nitrocellulose membrane (Invitrogen) as described above and analysed by immunoblotting.

For immunodetection of proteins, rabbit anti-GAL4-BD (sc-577, Santa Cruz Biotechnology, 1:2,000), goat anti-rabbit IgG-HRP (1:20,000, Sigma-Aldrich, A0545), mouse anti-Flag M2-HRP (1:60,000, Sigma-Aldrich) and rat anti-HA-HRP (clone 3F10, 1:1,1000, Roche) were used. Chemiluminescence was detected using the Immobilon western kit (Millipore) or the SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific) and the Syngene 680X EF GBOX imaging system (Syngene).

Protein production and purification

HMA domains

HMA domains were produced with a cleavable N-terminal 6xHis-MBP tag from E. coli BL21 (DE3) or BL21 (DE3) pLysS. 700 mL cell cultures were grown in LB media at 37 °C from a starting OD₆₀₀ of 0.05-0.07 to an OD₆₀₀ of 0.5-0.8. Protein production was induced by addition of IPTG (final concentration 1 mM) and cultures were grown for a further 14–16 hours at 20 °C. Cells were pelleted by centrifugation and the pellets stored at -70 °C. Thawed pellets were resuspended in 50 mM Tris-HCl pH 7, 300 mM NaCl, 30 mM imidazole supplemented with cOmplete EDTA-free protease inhibitor cocktail (Roche). Cells were lysed by sonication and the lysate clarified by centrifugation at 40,000 x g at 6 °C for 30 minutes. Subsequent chromatography steps were carried out with an ÄKTA Pure system (Cytiva) at 4 °C. The filtered (0.45 μm) supernatant was applied to a 5 ml HisTrap FF column (Cytiva) and, after washing with 50 mM Tris-HCl pH 7, 300 mM NaCl, 30 mM imidazole, bound protein was step-eluted with 50 mM Tris-HCl pH 7, 300 mM NaCl, 500 mM imidazole. The eluate was injected onto a Superdex 75 26/60 size exclusion chromatography column (Cytiva) pre-equilibrated in 50 mM Tris-HCl pH 7, 150 mM NaCl. Fractions containing the fusion protein were incubated with 3C protease overnight at 4 °C. HMA domains were separated from the 6xHis-MBP tag and GST-tagged 3C protease by injecting the protease-treated sample onto a 5 ml HisTrap FF column (Cytiva), 5 ml MBPTrap HP column (Cytiva), and 5 ml GSTrap HP column (Cytiva) connected in tandem and equilibrated in 50 mM Tris-HCl pH 7, 300 mM NaCl, 30 mM imidazole. Pooled fractions containing HMA domains were concentrated to < 8 ml then injected onto a Superdex 75 26/60 size exclusion chromatography column (Cytiva) pre-equilibrated in 50 mM Tris-HCl pH 7, 150 mM NaCl. Pooled fractions containing the purified HMA domains were concentrated. At each stage, fractions containing the HMA domain were determined by SDS-PAGE. Samples were combined with 5X SDS loading buffer (60 mM Tris-HCl pH 6.8, 25% glycerol, 4% SDS, 0.1% bromophenol blue, 10 mM DTT), heated at 95 °C for 5 minutes, then loaded onto a Bolt 10%, Bis-Tris gel (Invitrogen). After migration, gels were stained in InstantBlue Coomassie protein stain (Abcam). Final protein concentration was determined using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific), due to the extreme molar extinction coefficients of the proteins (0 M^{-1 cm-1} for OsHIPP21-HMA, 1490 M^{-1 cm-1} for OsHPP-HMA domains) at 280 nm. Proteins were stored at -80 °C.

MAX effectors

AVR-Pia, AVR-Pia-H3 and AVR1-CO39 were produced and purified as previously described [4,12,13]. Concentrations of MAX effectors were determined from molar extinction coefficients and absorbance at 280 nm.

Analytical size exclusion chromatography

Analytical size exclusion chromatography experiments were conducted using a Superdex 75 10/300 GL column (Cytiva) connected to an ÄKTA Pure system (Cytiva) at 4 °C. Running buffer was 50 mM Tris pH 7, 150 mM NaCl, 1 mM DTT for experiments with AVR1-CO39 and 20 mM sodium citrate pH 5.6, 150 mM NaCl, 1 mM DTT for experiments with AVR-Pia and AVR-Pia-H3. Running buffer was 50 mM Tris pH 7, 150 mM NaCl for analysis of OsHPP09^E16A-HMA and OsHPP09^E16R-HMA. To investigate whether OsHPP09-HMA forms a complex with the effectors, the two proteins were combined in a 1:1 molar ratio and incubated on ice for 1 h prior to analysis. Each protein was also analysed alone, at a concentration equivalent to that present in the mixture. For each experiment, 100 μl protein was injected onto the column and eluted at a flow rate of 0.5 ml/min. 500 μl fractions were collected for analysis by SDS-PAGE (as described in the protein purification section above).

Circular dichroism spectroscopy

Circular dichroism (CD) spectra were obtained using a Chirascan CD Spectrometer (Applied Photophysics). Proteins were dialysed into 10 mM sodium phosphate, pH 7.2 using Slide-A-Lyzer MINI dialysis devices (Thermo Fisher Scientific). 40 μl protein was used to fill a 0.1 mm cuvette. The sample compartment temperature was 20 °C. Spectra were obtained for wavelengths from 180 nm to 260 nm with step size of 1 nm, bandwidth of 2 nm and time-per-point of 2 s. Ten spectra were acquired for buffer and each sample. The average trace for buffer was subtracted from the average trace for each protein. Units were converted from millidegrees to mean residue molar ellipticity (MRME) using the Chirascan software.

Nuclear magnetic resonance titration

Samples of ¹⁵N-labelled AVR-Pia (40 μM) alone and complexed to unlabelled OsHPP09-HMA (1:0.5 or 1:1 molar ratio), or OsHPP09-HMA mutants or OsHIPP21-HMA, OsHPP10-HMA, and OsHPP11-HMA (1:1 molar ratio), were prepared in 20 mM sodium citrate, pH 5.6, 150 mM NaCl, and 1mM DTT, with addition of 10% D₂0 for the lock and 1 µM DSS as internal reference for the ¹H dimension and indirectly referenced for the ¹⁵N dimension [79]. The backbone (Hn, Nh, Cα) and Cß resonances of ¹⁵N,¹³C-labeled AVR-Pia complexed to OsHPP09-HMA (1:1 ratio) were assigned using three-dimensional (3D) HNCO, HNCA, HN(CO)CACB, HN(CA)CO and HNCACB experiments recorded at 305K on a Bruker Avance 800 MHz spectrometer equipped with a triple resonance (¹H, ¹⁵N, ¹³C) z-gradient cryo-probe, as described in detail for AVR-Pia alone in solution [13]. Spectra were processed using Topspin (v. 3.5pl6) and analysed with Cindy in-house software or CCPN [80] [analysis v 2.5.2].

For comparisons between free and OsHPP09-HMA-bound ¹⁵N-AVR-Pia, chemical shift perturbations (∆𝛿) were calculated using the equation [81]:

∆𝛿_𝐻 and ∆𝛿_𝑁 are the chemical shift differences measured in the proton dimension and nitrogen dimension, respectively, of 2D [¹H,¹⁵N] HSQC spectra recorded for AVR-Pia alone or in the presence of OsHPP09-HMA (1:1 ratio). ∆𝛿 greater than + 1σ or + 2σ were considered as significant or very significant, respectively, σ being the standard deviation measured on the distribution of chemical‐shift perturbations.

For comparisons between free ¹⁵N-AVR-Pia and ¹⁵N-AVR-Pia bound to OsHPP10-HMA, OsHPP11-HMA or OsHIPP21-HMA, peak heights were determined using CCPN [80] [analysis v 3] and used to calculate intensity ratios I/I0, where I and I0 represent the signal intensity for bound and free AVR-Pia, respectively.

Isothermal titration calorimetry (ITC)

ITC experiments were performed using a MicroCal PEAQ-ITC (Malvern Panalytical). Proteins were dialysed overnight into 20 mM sodium citrate pH 6.0, 150 mM NaCl, 1 mM TCEP. Experiments were carried out at 25 °C with a reference power of 5 μcal/s. The calorimetric cell was filled with purified HMA domain at 18 μM (OsHPP09-HMA), 35 μM (OsHIPP21-HMA and OsHPP11-HMA) or 60 μM (OsHPP10-HMA). This was titrated with AVR-Pia at a concentration 10X greater than that of the HMA domain (180 μM, 350 μM or 600 μM). Each experiment consisted of a single injection of 0.5 μL followed by 18 injections of 2 μL each at 150 s intervals with a stirring speed of 750 rpm. Data were processed with the MicroCal PEAQ-ITC analysis software (Malvern Panalytical). Experiments were carried out in triplicate.

Crystallisation of OsHPP09-HMA and AVR-Pia

Sitting drop, vapour diffusion crystallisation trials were set up in 96-well Swissci 3 lens crystallisation plates using dragonfly and mosquito dispensing robots (SPT Labtech). 100 nL of protein at 8.6 mg/ml was combined with 100 nL reservoir solution. Needle-like crystals were observed in multiple conditions in the commercial Structure Screen 1 + 2 (HT-96; Molecular Dimensions) and PEGs Suite (NeXtal) screens. Optimisation around these conditions yielded crystals suitable for diffraction studies. The X-ray diffraction data used to solve the structure were obtained from a crystal from 0.1 M MES pH 6.5, 0.2 M ammonium sulphate, 20% (w/v) PEG 4000. The crystal was mounted in a nylon loop and flash-frozen in liquid nitrogen prior to shipping to the European Synchrotron Radiation Facility (ESRF).

X-ray diffraction data collection and processing, and model refinement

X-ray diffraction data were collected on beamline MASSIF-1 (ID30A-1) at the ESRF. Data were processed with the EDNA autoprocessing pipeline [82]. The scaled, unmerged data file was passed to AIMLESS [83,84] for data reduction (implemented in CCP4i2 [85]). The AVR-Pia/OsHPP09-HMA structure was solved by molecular replacement with PHASER [86], using a monomer of AVR-Pia (PDB accession 6Q76, chain B) and a monomer of OsHPP09-HMA (modelled with ColabFold [54]). Iterative cycles of manual adjustment, refinement, and validation were carried out using COOT [87], REFMAC5 [88,89] and MolProbity [90,91]. Analysis of the interface was performed with qtPISA [57]. Interatomic distances were measured and structure superposition/RMSD calculations were performed with PyMOL v2.5.0 Open-Source [92]. Structure figures were prepared using the CCP4 molecular graphics (CCP4mg) software [93].

Structural modelling of AVR-Pia/HMA complexes

Structural modelling of AVR-Pia/HMA complexes was carried out with ColabFold v1.5.5 [54] (based on AlphaFold2 Multimer v3 [52,53]) and the AlphaFold3 web server (alphafoldserver.com; models generated between 2024-05-27 and 2024-07-02) [55]. For models generated with ColabFold, no template information from the PDB was used (template_mode: none) and number of recycles was set to 20 (num_recycles: auto). Five models were generated for each complex and relaxed using AMBER (num_relax: 5). Default settings were otherwise used. For models generated with AlphaFold3, default settings were used. At the time of use, AlphaFold3 did not support excluding template information from the PDB. Five models were generated for each complex using five different seed values. Interface analysis was carried out with qtPISA [57].

Supporting information

S1 Fig. AVR-Pia interacts with HMA domains from OsH(I)PPs.

Comprehensive pairwise Y2H analysis using AVR-Pia, MAX58, Pwl2, and AVR-PikD as prey, and a library of HMA domains from OsH(I)PPs as bait [49]. The left panel shows yeast growth on selective media, while the right panel identifies the rice H(I)PP proteins from which the HMA domains were derived. Diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH). Pictures were taken after 7 days of incubation. AD, activating domain; BD, binding domain; *, Autoactive BD construct leading to yeast growth regardless of the presence or identity of the AD construct; +++, strong yeast growth; ++, normal yeast growth, + , weak yeast growth; No HMA, proteins for which no HMA domain was identified by sequence analysis or structural prediction; Wrong annotation, constructs based on misannotated genes, resulting in missing or truncated HMA domains, thereby compromising Y2H assay.

https://doi.org/10.1371/journal.ppat.1014382.s001

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S2 Fig. Expression of BD-HMA constructs in haploid yeast.

Total yeast protein extracts from haploid yeast were separated on a 10% NuPAGE Bis-Tris gel and analysed by immunoblot using an anti-BD antibody to detect Gal4-BD fusion proteins with HMA domains from rice H(I)PPs. Blue asterisks indicate the band corresponding to the expected construct, while non-expressed constructs are marked in red. Protein loading is indicated by Ponceau S staining.

https://doi.org/10.1371/journal.ppat.1014382.s002

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S3 Fig. Mutation of the isoprenylation motif of OsHIPPs does not promote AVR-Pia interaction.

Y2H interaction analysis between AVR-Pia and either wildtype (WT) full-length (FL) OsHIPPs, including OsHIPP14, OsHIPP21, OsHIPP39 and OsHIPP41, or the corresponding FL OsHIPP isoprenylation mutants, in which the cysteine (C) residue was replaced by a non-prenylatable serine (S) residue. OsHPP09/AVR-Pia and was included as positive control. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to monitor growth (SD/-LW) or to assess protein-protein interactions (SD/-LWH). Photos were taken after 7 days of incubation. AD, activating domain; BD, binding domain. OsHIPP^C/S mutants: OsHIPP14^C187S, OsHIPP21^C133S, OsHIPP39^C190S, OsHIPP41^C152S.

https://doi.org/10.1371/journal.ppat.1014382.s003

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S4 Fig. Expression of bait and prey proteins in haploid yeast.

a Total yeast protein extracts from haploid yeast were separated on a 10 – 20% Tricine SDS-PAGE gel and analysed by immunoblot using an anti-BD antibody to detect Gal4-BD fusion proteins with either the heavy metal-associated (HMA) domain, full-length (FL) OsH(I)PPs and the OsHIPP21 isoprenylation mutant (C/S).Protein loading is indicated by Ponceau S staining (PS). b Total yeast protein extracts from haploid yeast were separated on a 10% NuPAGE Bis-Tris gel and analysed by immunoblot using an anti-BD antibody to detect Gal4-BD fusion proteins with either the HMA domain, FL OsHIPPs and their corresponding isoprenylation mutants (C/S). OsHIPP^C/S mutants: OsHIPP14^C187S, OsHIPP21^C133S, OsHIPP39^C190S, OsHIPP41^C152S. Protein loading is indicated by Ponceau S staining (PS). c Total yeast protein extracts from haploid yeast were separated on a NuPAGE Bis-Tris gel and analysed by immunoblot using an anti-HA antibody to detect Gal4-AD fusion proteins with MAX effectors. Protein loading is indicated by Ponceau S staining (PS). d Total yeast protein extracts from haploid yeast were separated on a 10 – 20% Tricine SDS-PAGE gel and analysed by immunoblot using an anti-BD antibody to detect Gal4-BD fusion proteins with FL OsHPP09 wildtype (WT) and mutant versions. Protein loading is indicated by Ponceau S staining (PS).

https://doi.org/10.1371/journal.ppat.1014382.s004

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S5 Fig. Amino acid alignment of OsHPP09, OsHPP10, OsHPP11, OsHIPP21 and the C-terminus of OsRGA5.

Amino acid alignment of full-length OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 and residues 995–1116 of OsRGA5, including the HMA domain and the C-terminal region of the protein which concludes with a putative CaaX isoprenylation motif (CSTM). Sequence alignment was carried out with Clustal Omega. The blue shaded box highlights the HMA domain, while the yellow shaded box highlights the CaaX motif present in OsHIPP21 and OsRGA5.

https://doi.org/10.1371/journal.ppat.1014382.s005

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S6 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 1).

Immunoblot analysis of transiently expressed proteins in N. benthamiana, including OsH(I)PPs N-terminally tagged with a 3xFlag epitope fused to the C-terminal part of luciferase (CLuc), and AVR-Pia and MAX58 N-terminally tagged with 3xHA and the N-terminal part of luciferase (NLuc). This experiment corresponds to replicate 1 of Fig 1. Detection was performed using anti-Flag and anti-HA antibodies. Membrane was stripped after anti-Flag detection and re-probed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S. staining (PS).

https://doi.org/10.1371/journal.ppat.1014382.s006

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S7 Fig. AlphaFold2 models do not differentiate between HMA domains which interact with AVR-Pia and those which do not.

AlphaFold2 (ColabFold v1.5.5) models of AVR-Pia in complex with HMA domains which a were experimentally shown to interact with AVR-Pia and b do not interact with AVR-Pia. Structure models are represented as ribbons and coloured by pLDDT score using the classical AlphaFold colour scheme as shown in the key. c Plots of ipTM, pTM and Multimer (0.8*iPTM + 0.2*pTM) scores for each of the five models generated for each HMA/AVR-Pia complex. Points indicate scores of individual models; green and purple lines indicate mean values for AVR-Pia-interacting and non-interacting HMA domains, respectively. d Comparison of interface parameters determined by qtPISA [57]) for the top ranked model of each HMA/AVR-Pia complex. Green and purple points indicate values for individual HMA/AVR-Pia models. The centre line of the box represents the median and the limits of the box represent the upper and lower quartiles. Whiskers extend to the smallest value within (Q1 − 1.5 × the interquartile range (IQR)) and the largest value within (Q3 + 1.5 × IQR).

https://doi.org/10.1371/journal.ppat.1014382.s007

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S8 Fig. AlphaFold3 models do not clearly differentiate between HMA domains which interact with AVR-Pia and those which do not.

AlphaFold3 models of AVR-Pia in complex with HMA domains which a were experimentally shown to interact with AVR-Pia and b do not interact with AVR-Pia. Structure models are represented as ribbons and coloured by pLDDT score using the classical AlphaFold colour scheme as shown in the key. c Plots of ipTM, pTM and Multimer (0.8*iPTM + 0.2*pTM) scores for each of the top ranked models from each of the five seeds used to model each HMA/AVR-Pia complex. Points indicate scores of individual models; green and purple lines indicate mean values for AVR-Pia-interacting and non-interacting HMA domains, respectively. d Comparison of interface parameters determined by qtPISA [57]) for the top ranked model (selected from models from all five seeds) of each HMA/AVR-Pia complex. Green and purple points indicate values for individual HMA/AVR-Pia models. The centre line of the box represents the median and the limits of the box represent the upper and lower quartiles. Whiskers extend to the smallest value within (Q1 − 1.5 × the interquartile range (IQR)) and the largest value within (Q3 + 1.5 × IQR).

https://doi.org/10.1371/journal.ppat.1014382.s008

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S9 Fig. Production and purification of OsHPP09-HMA.

a Size exclusion chromatography elution trace for 6xHis-MBP-OsHPP09-HMA (following the initial IMAC purification step). The SDS-PAGE gel shows fractions corresponding to the peak in the shaded area. b SDS-PAGE gel of pooled fractions following size exclusion chromatography before and after cleavage of the 6xHis-MBP tag with GST-tagged 3C protease. c SDS-PAGE gel showing flow-through, wash and elution fractions from tandem HisTrap, MBPTrap and GSTrap affinity columns. d Size exclusion chromatography elution trace for OsHPP09-HMA. OsHPP09-HMA absorbs light at 280 nm poorly (molar extinction coefficient of 1490 M^{-1 cm-1}). The SDS-PAGE gel shows fractions corresponding to the three peaks in the shaded areas. The peak elution volume of OsHPP09-HMA (193.9 ml) is consistent with dimerisation of the HMA domain.

https://doi.org/10.1371/journal.ppat.1014382.s009

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S10 Fig. Purification of the recombinant HMA domains of OsHPP10, OsHPP11 and OsHIPP21.

Representative size exclusion chromatography elution traces for a OsHPP10-HMA, b OsHPP11-HMA and c OsHIPP21-HMA. All three HMA domains absorb light at 280 nm poorly (molar extinction coefficients of 1490 M^{-1 cm-1}, 1490 M^{1 cm-1} and 0 M^{1 cm-1}, respectively) so the corresponding peaks are small for OsHPP10-HMA and OsHPP11-HMA and negligible for OsHIPP21). SDS-PAGE gels show fractions corresponding to the shaded areas of the trace. These fractions were pooled and concentrated to obtain the purified protein for subsequent analyses.

https://doi.org/10.1371/journal.ppat.1014382.s010

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S11 Fig. Purification of recombinant effectors AVR-Pia, AVR-Pia-H3 and AVR1-CO39.

Representative size exclusion chromatography elution traces for a AVR-Pia, b AVR-Pia-H3 and c AVR1-CO39. SDS-PAGE gels show fractions corresponding to the peaks in the shaded areas of the trace. These fractions were pooled and concentrated to obtain the purified protein for subsequent analyses.

https://doi.org/10.1371/journal.ppat.1014382.s011

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S12 Fig. The modelled dimerisation interface of OsHPP09-HMA overlaps with the modelled AVR-Pia binding surface.

Ribbon representation of the crystal structures of homodimers formed by the integrated HMA domains of a OsPikp-1-HMA and b OsRGA5-HMA and c the AlphaFold3 model of a homodimer of OsHPP09-HMA coloured by pLDDT score using the classical AlphaFold colour scheme.

https://doi.org/10.1371/journal.ppat.1014382.s012

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S13 Fig. Replicates of the ITC experiments presented in Fig 2.

Top panels show the raw thermograms obtained from titration of AVR-Pia into a solution containing the purified HMA domains. Central panels show the integrated heats (coloured dots) and binding isotherms fitted to a single site model (black line) using the MicroCal PEAQ-ITC analysis software (Malvern Panalytical). Bottom panels show the differences (coloured crosses) between the modelled and observed values (residuals).

https://doi.org/10.1371/journal.ppat.1014382.s013

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S14 Fig. Thermodynamic profiles from ITC experiments presented in Figs 2 and S8.

Bars represent the magnitude of the determined thermodynamic parameters ΔG (blue bar), ΔH (green bar) and -TΔS (red bar).

https://doi.org/10.1371/journal.ppat.1014382.s014

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S15 Fig. Controls for isothermal titration calorimetry experiments presented in Figs 2 and S13.

Representative raw thermograms from titration of buffer into a solution containing the purified HMA domains (top row) and titration of AVR-Pia into buffer (bottom row) at the concentrations used in the experiments presented in Figs 2 and S13.

https://doi.org/10.1371/journal.ppat.1014382.s015

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S16 Fig. AVR-Pia H3 differs from AVR-Pia in two amino acid positions.

a Multiple sequence alignment of AVR-Pia variants from Magnaporthe oryzae isolates described in [7]. Signal peptide is highlighted by a shaded grey box. Polymorphic residues 24 (F, S) and 46 (T, N) are indicated in a purple outlined box. Arrows above the alignment indicate secondary structure elements. b The polymorphic residues F24 and T46 are located at the HMA binding interface. Crystal structure of AVR-Pia in complex with Pikp-1-HMA (PDB 6Q76 [48]). AVR-Pia and Pikp-1-HMA are represented as gold and teal ribbons, respectively, with the side chains of AVR-Pia T46 and F24S shown in purple.

https://doi.org/10.1371/journal.ppat.1014382.s016

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S17 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 3).

Immunoblot analysis of transiently expressed proteins in N. benthamiana, including OsH(I)PPs and OsRGA5_Cter (883 – 1116 aa) N-terminally tagged with a 3xFlag epitope fused to the C-terminal part of luciferase (CLuc), and AVR-Pia-H3 N-terminally tagged with 3xHA and the N-terminal part of luciferase (NLuc). This experiment corresponds to replicate 2 of Fig 3. Detection was performed using anti-Flag and anti-HA antibodies. Membrane was stripped after anti-Flag detection and re-probed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S. staining (PS). S18 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 4).

https://doi.org/10.1371/journal.ppat.1014382.s017

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S18 Fig. Immunoblot analysis of transiently expressed proteins in N. benthamiana, including OsH(I)PPs and OsRGA5_Cter (883 – 1116 aa) N-terminally tagged with a 3xFlag epitope fused to the C-terminal part of luciferase (CLuc), and AVR1-CO39 N-terminally tagged with 3xHA and the N-terminal part of luciferase (NLuc).

This experiment corresponds to replicate 1 of Fig 4. Detection was performed using anti-Flag and anti-HA antibodies. Membrane was stripped after anti-Flag detection and re-probed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S. staining (PS).

https://doi.org/10.1371/journal.ppat.1014382.s018

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S19 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 5).

Immunoblot analysis of transiently expressed proteins in N. benthamiana, including OsH(I)PPs N-terminally tagged with a 3xFlag epitope fused to the C-terminal part of luciferase (CLuc), and AVR-Pia, AVR-PikC, AVR-PikD and Pwl2 N-terminally tagged with 3xHA and the N-terminal part of luciferase (NLuc). This experiment corresponds to replicate 1 of Fig 5. Detection was performed using anti-Flag and anti-HA antibodies. Membrane was stripped after anti-Flag detection and re-probed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S. staining (PS).

https://doi.org/10.1371/journal.ppat.1014382.s019

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S20 Fig. Overlay of HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (purple cross-peaks) of unlabelled OsHPP09-HMA (40 μM).

Grey cross-peak labels are used for residues with little or no chemical shift perturbation. For substantial chemical shifts, black cross-peak labels correspond to spectra for ¹⁵N-AVR-Pia alone (previously assigned [4]) and red cross-peak labels correspond to spectra for ¹⁵N-AVR-Pia in the presence of OsHPP09-HMA.

https://doi.org/10.1371/journal.ppat.1014382.s020

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S21 Fig. The AVR-Pia/OsHPP09-HMA complex is in slow exchange.

Cross-peaks corresponding to residues G30, I44, E56 and Y85 extracted from [¹H,¹⁵N] HSQC spectra from NMR titrations performed with ¹⁵N-labelled AVR-Pia and OsHPP09-HMA at molar ratios of 1:0 (free AVR-Pia; orange cross-peaks), 1:0.5 (intermediate; red cross-peaks) and 1:1 (bound AVR-Pia; purple cross-peaks). The final column shows the overlay of the different spectra.

https://doi.org/10.1371/journal.ppat.1014382.s021

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S22 Fig. The OsHPP09-HMA binding interface involves residues in β2 and β3 of AVR-Pia.

a Barplot of ¹H/¹⁵N chemical shift perturbations (Δδ) for the amide groups of AVR-Pia. Dashed lines represent Δδ thresholds of + 1σ and + 2σ. Significant chemical shift variations (> + 1σ or> + 2σ) are indicated in red and purple, respectively. b Residues giving significant chemical shift variations of> + 1σ or> + 2σ represented in red or purple, respectively, on the crystal structure (PDB 9RSV) of AVR-Pia (surface representation) bound to OsHPP09-HMA (teal ribbon representation). The two views are rotated by 90° as indicated.

https://doi.org/10.1371/journal.ppat.1014382.s022

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S23 Fig. AVR-Pia interacts with the HMA domains of OsHPP10, OsHPP11 and OsHIPP21 through a similar surface.

a-c Residue-resolved NMR signal intensity ratios I/I0, where I0 represents the signal intensity for free AVR-Pia and I represents the signal intensity for AVR-Pia bound to OsHPP10-HMA (a), OsHPP11-HMA (b) or OsHIPP21-HMA (c). The boundaries of the six β-strands of the AVR-Pia MAX fold are shown at the bottom of the plot (grey arrows). Bars are coloured grey for values of I/I0 > x̄, gold if x̄ > I/I0> (x̄ - σ), and pink if I/I0 <(x̄ - σ). Red dashed lines indicate I/I0 thresholds x̄ (upper) and x̄-σ (lower). d-f Ribbon representation of AlphaFold3 models of the complexes between AVR-Pia and OsHPP10-HMA (d), OsHPP11-HMA (e) or OsHIPP21-HMA (f). AVR-Pia residues are coloured by I/I0. g-i Overlay of HSQC spectra of 40 μM ¹⁵N-AVR-Pia alone (pink) and presence of 40 μM unlabelled OsHPP10-HMA (green) (g), OsHPP11-HMA (grey) (h) or OsHIPP21-HMA (cyan) (i). Upon complex formation with AVR-Pia, the majority of peaks for residues in β2 and β3 were broadened out beyond detection, whereas residues on the opposite face of AVR-Pia were less affected.

https://doi.org/10.1371/journal.ppat.1014382.s023

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S24 Fig. Comparison of binding interfaces in the crystal structures of AVR-Pia in complex with OsHPP09-HMA (PDB accession code 9RSV), AVR-Pia/Pikp-1-HMA (6Q76) [48] and AVR1-CO39/RGA5-HMA (5ZNG) [12]. a Global interface of the effector/HMA complex.

Structures are represented as gold (AVR-Pia), teal (OsHPP09-HMA), brown (AVR-Pia), pale blue (Pikp-1-HMA), orange (AVR1-CO39) and green (RGA5-HMA) ribbons with relevant secondary structure elements indicated. b Hydrogen bonds between residues in β2 of the effector and β2 of the HMA domain. Main chain atoms are represented as cylinders (only the main chain, and not the side chains, are included for clarity). Hydrogen bonds are represented as black dashed lines with lengths (determined by qtPISA [57]) indicated. c-f Comparison of residues involved in forming intermolecular contacts at the effector/HMA interface. Structures are presented in ribbon representation with relevant residues shown as cylinders. Hydrogen bonds are represented as black dashed lines with lengths (determined by qtPISA [57]) indicated. Water molecules are represented as red spheres.

https://doi.org/10.1371/journal.ppat.1014382.s024

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S25 Fig. Similarities and differences between AlphaFold2 and AlphaFold3 models and the experimental structure of the AVR-Pia/OsHPP09-HMA complex.

a Hydrogen bonds between residues in β2 of the effector and β2 of the HMA domain. Structures are represented as gold (AVR-Pia, experimental structure), teal (OsHPP09-HMA, experimental structure), brown (AVR-Pia, AlphaFold2 model), dark teal (OsHPP09-HMA, AlphaFold2 model), pale brown (AVR-Pia, AlphaFold3 model) and pale blue (OsHPP09-HMA, AlphaFold3 model). Main chain atoms are represented as cylinders (only the main chain, and not the side chains, are included for clarity). Hydrogen bonds are represented as black dashed lines with lengths (determined by qtPISA [57]) indicated. Where hydrogen bonds are present in the OsHPP09-HMA/AVR-Pia crystal structure but the corresponding atoms in the AlphaFold models are separated by a distance greater than 3.5 Å, the distance (measured with PyMOL [92]) is indicated in brackets. b Position of the sidechain of AVR-Pia^R43 in the crystal structure and AlphaFold models. c Position of the sidechain of OsHPP09-HMA^E16 in the crystal structure and AlphaFold models.

https://doi.org/10.1371/journal.ppat.1014382.s025

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S26 Fig. Comparison of intermolecular hydrogen bonding between AVR-Pia and OsHPP09-HMA in the crystal structure (PDB accession code 9RSV) and in the AlphaFold2 and AlphaFold3 models.

2D protein-protein interaction diagrams adapted from the output of the DimPlot module of LigPlot+ (v2.3.1; [95,96]). Hydrogen bonds present in the qtPISA [57] output but absent from the HBPLUS output (used by LigPlot) were added manually. Intermolecular hydrogen bonds between atoms in AVR-Pia and OsHPP09-HMA are represented as green dashed lines, while hydrogen bonds involving a water molecule are represented as purple dashed lines. Water molecules are indicated by sky blue circles. Bond lengths are indicated.

https://doi.org/10.1371/journal.ppat.1014382.s026

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S27 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 7).

Immunoblot analysis of transiently expressed proteins in N. benthamiana, including OsHPP09 wildtype (WT) and mutant versions N-terminally tagged with a 3xFlag epitope fused to the C-terminal part of luciferase (CLuc), and AVR-Pia N-terminally tagged with 3xHA and the N-terminal part of luciferase (NLuc). This experiment corresponds to replicate 2 of Fig 7. Detection was performed using anti-Flag and anti-HA antibodies. Membrane was stripped after anti-Flag detection and re-probed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S. staining (PS). Dashed lines between bands indicate that lanes were cut from the same blot but were non-adjacent.

https://doi.org/10.1371/journal.ppat.1014382.s027

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S28 Fig. Purification of the recombinant HMA domain of OsHPP09 with D33H, E16A, E16R or I35K mutations.

Representative size exclusion chromatography elution traces for a OsHPP09-HMA^D33H, b OsHPP09-HMA^E16A, c OsHPP09-HMA^E16R and d OsHPP09-HMA^I35K. The HMA domains absorb light at 280 nm poorly (molar extinction coefficient of 1490 M^{-1 cm-1}) so the corresponding peaks are small. SDS-PAGE gels show fractions corresponding to the shaded areas of the trace. These fractions were pooled and concentrated to obtain the purified protein for subsequent analyses.

https://doi.org/10.1371/journal.ppat.1014382.s028

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S29 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (turquoise cross-peaks) of unlabelled OsHPP09^D33H-HMA (40 μM).

Cross-peak labels correspond to the ¹⁵N-AVR-Pia spectra (previously assigned [4]). Zoom inset panels show cross-peaks corresponding to residues G30, I44, E56 and Y85 to illustrate the chemical shift perturbations between the free (¹⁵N-AVR-Pia alone) and bound (¹⁵N-AVR-Pia with OsHPP09^D33H-HMA) as presented in Fig 7. Zoomed areas are indicated by boxes on the spectra.

https://doi.org/10.1371/journal.ppat.1014382.s029

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S30 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (blue cross-peaks) of unlabelled OsHPP09^E16A-HMA (40 μM).

Cross-peak labels correspond to the ¹⁵N-AVR-Pia spectra (previously assigned [4]). Zoom inset panels show cross-peaks corresponding to residues G30, I44, E56 and Y85 to illustrate the chemical shift perturbations between the free (¹⁵N-AVR-Pia alone) and bound (¹⁵N-AVR-Pia with OsHPP09^E16A-HMA) as presented in Fig 7. Zoomed areas are indicated by boxes on the spectra.

https://doi.org/10.1371/journal.ppat.1014382.s030

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S31 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (pale blue cross-peaks) of unlabelled OsHPP09^E16R-HMA (40 μM).

Cross-peak labels correspond to the ¹⁵N-AVR-Pia spectra (previously assigned [4]). Zoom inset panels show cross-peaks corresponding to residues G30, I44, E56 and Y85 to illustrate the chemical shift perturbations between the free (¹⁵N-AVR-Pia alone) and bound (¹⁵N-AVR-Pia with OsHPP09^E16R-HMA) as presented in Fig 7. Zoomed areas are indicated by boxes on the spectra.

https://doi.org/10.1371/journal.ppat.1014382.s031

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S32 Fig. Mutation of E16 does not affect dimerization of OsHPP09-HMA.

a Ribbon representation of the AlphaFold3 model (presented in S12 Fig coloured by pLDDT score) of a homodimer of OsHPP09-HMA (teal). Residues forming hydrogen bonds with the opposite protomer are represented as cylinders and hydrogen bonds are represented as black dashed lines. b Analytical gel filtration traces obtained from injection of OsHPP09-HMA^E16A (top panel) or OsHPP09-HMA^E16R (bottom panel). Significant peaks are indicated by coloured dashed lines with elution volume labelled. SDS-PAGE gel inserts show fractions from peak elution volumes indicated by grey shaded regions. OsHPP09-HMA^E16A and OsHPP09-HMA^E16R absorb light at 280 nm poorly (molar extinction coefficient: 1490 M^{-1 cm-1}) so the corresponding peak is small.

https://doi.org/10.1371/journal.ppat.1014382.s032

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S33 Fig. The residue at position 16 in OsHPP09 and OsHPP10 determines strength of binding to AVR-Pia.

a Y2H assay testing interactions between AVR-Pia, AVR1-CO39, and AVR-PikC (all lacking signal peptide, ΔSP) and the HMA domains or full-length (FL) forms of OsHPP09 and OsHPP10. Both wild-type proteins and reciprocal mutants at position 16 were analysed, including OsHPP09^E16D and OsHPP10^D16E. Serial dilutions of diploid yeast were spotted onto synthetic defined (SD) media to assess growth (SD/ − LW) and protein–protein interactions (SD/ − LWH and SD/ − LWH supplemented with 0.5 mM 3-amino-1,2,4-triazole [3-AT]). Images were captured after 7 days of incubation. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. b Total protein extracts from haploid yeast cells were separated on a 10–20% Tricine SDS–PAGE gel and analysed by immunoblot using an anti-BD antibody to detect Gal4-BD fusion proteins containing wild-type or mutant HMA domains of OsHPP09 and OsHPP10, or the corresponding full-length (FL) proteins. Protein loading is indicated by Ponceau S staining (PS).

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S34 Fig. Mutation of OsHPP09 E16, OsHPP10 D16 and OsHIPP21 E15 reduces AVR-Pia binding.

a Split luciferase complementation assays assessing interaction between AVR-Pia (ΔSP; fused to the N-terminal luciferase fragment, 3 × HA:NLuc:AVR-Pia) and full-length H(I)PP proteins carrying mutations at the position equivalent to OsHPP09 E16 (fused to the C-terminal luciferase fragment, 3 × Flag:CLuc:OsH(I)PPs). Co-expression of AVR-Pia with wild-type OsH(I)PPs served as a positive control, whereas co-expression with OsHIPP43 served as a negative control. Leaves infiltrated with P19 alone were used as background (BG) control. Box plots display the median (line), mean (cross), and interquartile range (box limits); whiskers extend to the most extreme values within 1.5 × the interquartile range, with outliers shown individually. Three independent experiments were performed, each with n = 6 per combination. Data points are shown with symbols corresponding to each replicate. Different letters indicate statistically significant differences based on pairwise Wilcoxon tests (α = 0.05). b Immunoblot analysis of transiently expressed proteins in N. benthamiana. Full-length OsH(I)PPs were fused to a C-terminal 3 × Flag tag and the C-terminal luciferase fragment (CLuc), while AVR-Pia was fused N-terminally to 3 × HA and the N-terminal luciferase fragment (NLuc). Shown is replicate 3. Proteins were detected using anti-Flag and anti-HA antibodies; the membrane was stripped after anti-Flag detection and reprobed with anti-HA. Protein loading is indicated by the Rubisco band visualized by Ponceau S staining (PS).

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S35 Fig. Comparison of binding interfaces in the crystal structures of MAX effectors bound to HMA domains of H(I)PPs.

The structures of AVR-Pia in complex with OsHPP09-HMA (PDB accession code 9RSV), AVR-PikF in complex with OsHIPP19-HMA (PDB accession code 7B1I [8]) and Pwl2 in complex with OsHIPP43-HMA (PDB accession code 8R7A [9]) are represented as gold (AVR-Pia), teal (OsHPP09-HMA), brown (Pwl2), green (OsHIPP43-HMA), pink (AVR-PikF) and grey (OsHIPP19-HMA) ribbons with relevant secondary structure elements indicated. Superposition of MAX effectors (left) and HMA domains (right) was carried out in CCP4mg [93] using secondary structure matching.

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S36 Fig. Comparison of binding interfaces in the crystal structure of AVR-Pia in complex with OsHPP09-HMA (PDB accession code 9RSV) and the AlphaFold3 models of AVR-Pia/OsHPP10-HMA, AVR-Pia/OsHPP11-HMA and AVR-Pia/OsHIPP21-HMA (global structures coloured by pLDDT score are presented in S8 Fig).

a Hydrogen bonds between residues in β2 of the effector and β2 of the HMA domain. Structures are represented as gold (AVR-Pia), teal (OsHPP09-HMA), green (OsHPP10-HMA), purple (OsHPP11-HMA) and turquoise (OsHIPP21-HMA) ribbons Main chain atoms are represented as cylinders (only the main chain, and not the side chains, are included for clarity). Hydrogen bonds are represented as black dashed lines with lengths (determined by qtPISA [57]) indicated. b-e Comparison of residues involved in forming intermolecular contacts at the effector/HMA interface in the crystal structure of AVR-Pia/OsHPP09-HMA with the corresponding residues in OsHPP10-HMA, OsHPP11-HMA and OsHIPP21-HMA. Structures are presented in ribbon representation with relevant residues shown as cylinders. Hydrogen bonds are represented as black dashed lines with lengths (determined by qtPISA [92]) indicated. Where hydrogen bonds are present in the OsHPP09-HMA/AVR-Pia crystal structure but the corresponding atoms in the AlphaFold3 models are separated by a distance greater than 3.5 Å, the distance (measured with PyMOL [92]) is indicated in brackets.

https://doi.org/10.1371/journal.ppat.1014382.s036

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S1 Table. Amino acid sequences and MSU/RGAP and RAP-DB/IRGSP identifiers for the H(I)PPs used in the present study.

https://doi.org/10.1371/journal.ppat.1014382.s037

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S2 Table. Rice proteins interacting with AVR-Pia in the Y2H screen.

Results of a Y2H screen using BD:AVR-Pia as bait and a cDNA library from the rice cultivar CO39. The number of positive clones with insert sequences (cloned in frame with Gal4 AD) corresponding to the designated proteins are shown. * Potential false positives found in >50% of the Y2H screens performed in the lab using various bait proteins.

https://doi.org/10.1371/journal.ppat.1014382.s038

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S3 Table. Thermodynamic parameters obtained from ITC experiments.

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S4 Table. X-ray data collection and refinement statistics for OsHPP09-HMA/ AVR-Pia.

https://doi.org/10.1371/journal.ppat.1014382.s040

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S5 Table. Comparison of the binding interfaces of complexes between HMA domains and MAX effectors.

Interface analysis was performed using qtPISA [57].

https://doi.org/10.1371/journal.ppat.1014382.s041

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S6 Table. List of primers used in this study.

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S7 Table. List of constructs generated in this study.

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Acknowledgments

We thank Mark Youles and Mark Banfield for generating and providing the pOPIN-GG vector and tag modules, Laurent Deslandes for providing the split-luciferase vectors, and Florian Veillet for providing the pFV79 construct. We thank Thorsten Langner for providing the Y2H HMA library. We thank Julie Noell for assistance with protein purification, Corinne Lionne and Manon Mallet for helpful discussions about ITC methods, and Christian Roumestand for advice about NMR data. We thank Sebastien Ribeiro, Laura Mathieu and Vincent Chochois for valuable discussions and support with data analysis. This work benefited from access to the NMR and RX facilities of the Integrated Biophysics and Structural Biology Platform (PIBBS) of the CBS. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities. We thank the staff of the ESRF and EMBL Grenoble for assistance in using beamline ID30-A1 under proposal number mx-2409.

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Figures

Abstract

Author summary

Introduction

Results

AVR-Pia interacts with rice H(I)PPs in Y2H screening

AVR-Pia interacts with rice H(I)PPs in planta

AlphaFold Multimer models cannot differentiate between HMA domains which are bound by AVR-Pia and those which are not

AVR-Pia interacts with the HMA domains of OsHPP09, OsHPP10, OsHPP11 and OsHIPP21 in vitro with varying affinities

AVR-Pia-H3 does not interact with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21

AVR1-CO39 does not interact with OsHPP09, OsHPP10, OsHPP11 and OsHIPP21

AVR-Pia interacts with a distinct group of H(I)PPs to AVR-Pik and Pwl2

AVR-Pia interacts with the four HMA domains through a similar surface

The crystal structure of the AVR-Pia/OsHPP09-HMA complex reveals interface contacts underpinning high affinity binding

Replacement of OsHPP09E16 with alanine or arginine disrupts AVR-Pia binding

Residues corresponding to OsHPP09E16 in OsHPP10 and OsHIPP21 are important for AVR-Pia binding

Discussion

Methods

Cloning

Yeast two-hybrid

Yeast two-hybrid cDNA library screening

Agrobacterium-mediated transient expression in N. benthamiana

Split luciferase assay

Protein extraction and Western blotting

Protein production and purification

HMA domains

MAX effectors

Analytical size exclusion chromatography

Circular dichroism spectroscopy

Nuclear magnetic resonance titration

Isothermal titration calorimetry (ITC)

Crystallisation of OsHPP09-HMA and AVR-Pia

X-ray diffraction data collection and processing, and model refinement

Structural modelling of AVR-Pia/HMA complexes

Supporting information

S1 Fig. AVR-Pia interacts with HMA domains from OsH(I)PPs.

S2 Fig. Expression of BD-HMA constructs in haploid yeast.

S3 Fig. Mutation of the isoprenylation motif of OsHIPPs does not promote AVR-Pia interaction.

S4 Fig. Expression of bait and prey proteins in haploid yeast.

S5 Fig. Amino acid alignment of OsHPP09, OsHPP10, OsHPP11, OsHIPP21 and the C-terminus of OsRGA5.

S6 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 1).

S7 Fig. AlphaFold2 models do not differentiate between HMA domains which interact with AVR-Pia and those which do not.

S8 Fig. AlphaFold3 models do not clearly differentiate between HMA domains which interact with AVR-Pia and those which do not.

S9 Fig. Production and purification of OsHPP09-HMA.

S10 Fig. Purification of the recombinant HMA domains of OsHPP10, OsHPP11 and OsHIPP21.

S11 Fig. Purification of recombinant effectors AVR-Pia, AVR-Pia-H3 and AVR1-CO39.

S12 Fig. The modelled dimerisation interface of OsHPP09-HMA overlaps with the modelled AVR-Pia binding surface.

S13 Fig. Replicates of the ITC experiments presented in Fig 2.

S14 Fig. Thermodynamic profiles from ITC experiments presented in Figs 2 and S8.

S15 Fig. Controls for isothermal titration calorimetry experiments presented in Figs 2 and S13.

S16 Fig. AVR-Pia H3 differs from AVR-Pia in two amino acid positions.

S17 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 3).

S19 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 5).

S20 Fig. Overlay of HSQC spectra of 15N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (purple cross-peaks) of unlabelled OsHPP09-HMA (40 μM).

S21 Fig. The AVR-Pia/OsHPP09-HMA complex is in slow exchange.

S22 Fig. The OsHPP09-HMA binding interface involves residues in β2 and β3 of AVR-Pia.

S23 Fig. AVR-Pia interacts with the HMA domains of OsHPP10, OsHPP11 and OsHIPP21 through a similar surface.

S24 Fig. Comparison of binding interfaces in the crystal structures of AVR-Pia in complex with OsHPP09-HMA (PDB accession code 9RSV), AVR-Pia/Pikp-1-HMA (6Q76) [48] and AVR1-CO39/RGA5-HMA (5ZNG) [12]. a Global interface of the effector/HMA complex.

S25 Fig. Similarities and differences between AlphaFold2 and AlphaFold3 models and the experimental structure of the AVR-Pia/OsHPP09-HMA complex.

S26 Fig. Comparison of intermolecular hydrogen bonding between AVR-Pia and OsHPP09-HMA in the crystal structure (PDB accession code 9RSV) and in the AlphaFold2 and AlphaFold3 models.

S27 Fig. Expression of CLuc- and NLuc fusion proteins (related to Fig 7).

S28 Fig. Purification of the recombinant HMA domain of OsHPP09 with D33H, E16A, E16R or I35K mutations.

S29 Fig. HSQC spectra of 15N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (turquoise cross-peaks) of unlabelled OsHPP09D33H-HMA (40 μM).

S30 Fig. HSQC spectra of 15N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (blue cross-peaks) of unlabelled OsHPP09E16A-HMA (40 μM).

S31 Fig. HSQC spectra of 15N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (pale blue cross-peaks) of unlabelled OsHPP09E16R-HMA (40 μM).

S32 Fig. Mutation of E16 does not affect dimerization of OsHPP09-HMA.

S33 Fig. The residue at position 16 in OsHPP09 and OsHPP10 determines strength of binding to AVR-Pia.

S34 Fig. Mutation of OsHPP09 E16, OsHPP10 D16 and OsHIPP21 E15 reduces AVR-Pia binding.

S35 Fig. Comparison of binding interfaces in the crystal structures of MAX effectors bound to HMA domains of H(I)PPs.

S36 Fig. Comparison of binding interfaces in the crystal structure of AVR-Pia in complex with OsHPP09-HMA (PDB accession code 9RSV) and the AlphaFold3 models of AVR-Pia/OsHPP10-HMA, AVR-Pia/OsHPP11-HMA and AVR-Pia/OsHIPP21-HMA (global structures coloured by pLDDT score are presented in S8 Fig).

S1 Table. Amino acid sequences and MSU/RGAP and RAP-DB/IRGSP identifiers for the H(I)PPs used in the present study.

S2 Table. Rice proteins interacting with AVR-Pia in the Y2H screen.

S3 Table. Thermodynamic parameters obtained from ITC experiments.

S4 Table. X-ray data collection and refinement statistics for OsHPP09-HMA/ AVR-Pia.

S5 Table. Comparison of the binding interfaces of complexes between HMA domains and MAX effectors.

S6 Table. List of primers used in this study.

S7 Table. List of constructs generated in this study.

Acknowledgments

References

Replacement of OsHPP09^E16 with alanine or arginine disrupts AVR-Pia binding

Residues corresponding to OsHPP09^E16 in OsHPP10 and OsHIPP21 are important for AVR-Pia binding

S20 Fig. Overlay of HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (purple cross-peaks) of unlabelled OsHPP09-HMA (40 μM).

S29 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (turquoise cross-peaks) of unlabelled OsHPP09^D33H-HMA (40 μM).

S30 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (blue cross-peaks) of unlabelled OsHPP09^E16A-HMA (40 μM).

S31 Fig. HSQC spectra of ¹⁵N-AVR-Pia (40 μM) in the absence (orange cross-peaks) and presence (pale blue cross-peaks) of unlabelled OsHPP09^E16R-HMA (40 μM).