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

Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik from rice blast fungus Magnaporthe oryzae is a pair of NLR Pik-1 and Pik-2. contains noncanonical integrated heavy-metal-associated (HMA) domain, directly binds activate defenses. targets are also HMA-domain-containing namely isoprenylated proteins (HIPPs) (HPPs). Here, we demonstrate that one these interacts with wider set variants compared HMA domains. We define biochemical structural basis interaction between OsHIPP19 compare formed domain Pik-1. Using analytical gel filtration surface plasmon resonance, show multiple variants, including stealthy AVR-PikC AVR-PikF, do not interact any characterized alleles, bind nanomolar affinity. crystal structure in complex AVR-PikF reveals differences at interface underpin high-affinity binding OsHIPP19-HMA than achieved Our results provide foundation for engineering extend currently unrecognized expand disease resistance divergent pathogen strains. Phytopathogens constrain crop production threaten global food security. filamentous ascomycete causative agent disease, annually destroys enough feed upward 200 million people year (1Wilson R.A. Talbot N.J. Under pressure: Investigating biology infection oryzae.Nat. Rev. Microbiol. 2009; 7: 185-195Crossref PubMed Scopus (564) Google Scholar, 2Dean R. Van Kan J.A.L. Pretorius Z.A. Hammond-Kosack K.E. Di Pietro A. Spanu P.D. Rudd J.J. Dickman M. Kahmann Ellis J. Foster G.D. top 10 fungal molecular pathology.Mol. Plant Pathol. 2012; 13: 414-430Crossref (1779) Scholar). found all major rice-growing regions around world (3Wang X. Lee S. Wang Ma Bianco T. Ji Y. Current advances on genetic disease.in: Yan W. Bao Rice - Germplasm, Genetics Improvement. IntechOpen, London, UK2014: 195-217Crossref Scholar), severe epidemics cause total loss (2Dean 4Pennisi E. Armed dangerous.Science. 2010; 327: 804-805Crossref (224) In addition rice, strains infect other staple crops such as wheat, barley millet, various wild grass species (5Kato H. Yamamoto Yamaguchi-Ozaki Pathogenicity, mating ability DNA restriction fragment length polymorphisms Pyricularia populations isolated Gramineae, Bambusideae Zingiberaceae plants.J. Gen. 2000; 66: 30-47Crossref 6Yoshida K. Saunders D.G. Mitsuoka C. Natsume Kosugi Saitoh Inoue Chuma I. Tosa Cano L.M. Kamoun Terauchi Host specialization associated dynamic gain genes linked transposable elements.BMC Genomics. 2016; 18: 370Crossref (81) During infection, phytopathogens deliver apoplast inside cells. These effectors function diverse ways suppress immunity endogenous processes create favorable conditions colonization. While many candidates have been identified cloned (7Petit-Houdenot Langner Harant Win A clone resource share sequence similarities across host-specific lineages.Mol. Microbe Interact. 2020; 33: 1032-1035Crossref (4) only few functionally (8Park C.-H. Chen Shirsekar G. Zhou B. Khang C.H. Songkumarn P. Afzal A.J. Ning Bellizzi Valent G.-L. AvrPiz-t RING E3 ubiquitin ligase APIP6 pathogen-associated pattern–triggered rice.Plant Cell. 24: 4748Crossref (287) 9Park Xie Shi Suttiviriya Umemura APIP10 connects receptor Piz-t rice.PLoS Pathog. 12e1005529Crossref (74) 10Tang Shu Dong Zhang Wu D. Nup98 homolog APIP12 targeted involved basal against oryzae.Rice (N. Y.). 2017; 10: 5Crossref (29) 11Wang He F. Fan Jiang N. Hu G.L. Immunity suppression effector-triggered necrosis.Curr. Biol. 26: 2399-2411Abstract Full Text PDF (47) 12Orbach M.J. Farrall L. Sweigard J.A. Chumley F.G. telomeric avirulence gene determines efficacy Pi-ta.Plant 12: 2019-2032Crossref (373) 13Jia McAdams S.A. Bryan G.T. Hershey H.P. Direct products confers resistance.EMBO 19: 4004-4014Crossref (793) For most described date, mechanism they virulence remains unknown. presence some cells detected specific nucleotide-binding, containing (NLRs) (14Jones J.D.G. Vance R.E. Dangl J.L. Intracellular innate surveillance devices plants animals.Science. 354aaf6395Crossref (438) NLRs instigate defensive signaling pathways following perception protein, either through direct or monitoring state intermediate protein (15Jones system.Nature. 2006; 444: 323-329Crossref (7505) 16van der Hoorn R.A.L. From Guard Decoy: new model effectors.Plant 2008; 20: 2009-2017Crossref (453) 17Dangl Jones J.D. defence responses infection.Nature. 2001; 411: 826-833Crossref (2853) canonical comprises C-terminal central NB-ARC N-terminal coiled-coil (CC CCR (18Collier S.M. Hamel L.P. Moffett Cell death mediated domains unique highly conserved class NB-LRR protein.Mol. 2011; 918-931Crossref (190) Scholar)) Toll/interleukin-1 (TIR) (19Pan Q. Wendel Fluhr Divergent evolution NBS-LRR homologues dicot cereal genomes.J. Mol. Evol. 50: 203-213Crossref (334) 20Takken F.L.W. Albrecht Tameling W.I.L. Resistance proteins: Molecular switches defence.Curr. Opin. 9: 383-390Crossref (289) 21Tameling Elzinga S.D.J. Darmin P.S. Vossen J.H. Takken Haring M.A. Cornelissen B.J.C. tomato R I-2 Mi-1 functional ATP ATPase activity.Plant 2002; 14: 2929-2939Crossref (302) Recent studies different (22Cesari Thilliez Ribot Chalvon V. Michel Jauneau Rivas Alaux Kanzaki Okuyama Morel J.-B. Fournier Tharreau Kroj RGA4/RGA5 recognizes AVR-Pia AVR1-CO39 binding.Plant 2013; 25: 1463Crossref (259) 23Sarris P.F. Cevik Dagdas Krasileva K.V. Comparative analysis architectures uncovers likely pathogens.BMC 1-19Crossref (143) 24Kroj Chanclud Michel-Romiti Grand Integration decoy derived into receptors widespread.New Phytol. 210: 618-626Crossref (125) (IDs) thought their evolutionary origins (25Cesari Bernoux Moncuquet Dodds P.N. novel pairs: “integrated decoy” hypothesis.Front. Sci. 2014; 5: 606Crossref (197) 26Wu Banfield “sensor domains” More decoys?.Front. 2015; 6: 134Crossref (50) By acting substrate interactor, enable detect trigger immunity. paired CC-NLRs Pik-2 genetically cooperate response (27Ashikawa Hayashi Yamane Kanamori Matsumoto Ono Yano Two adjacent site-leucine-rich required confer Pikm-specific resistance.Genetics. 180: 2267-2276Crossref (242) 28Kanzaki Yoshida Fujisaki Hirabuchi Arms race co-evolution Pik driven physical interactions.Plant 72: 894-907Crossref (162) 29Maqbool Franceschetti Stevenson C.E.M. Uemura Structural recognition receptor.Elife. 4e08709Crossref (130) CC (29Maqbool Previous work demonstrated detection Five alleles 30Yuan Zhai Zeng Xu Lin Pan Pik-p closely CC-NBS-LRR genes.Theor. Appl. Genet. 122: 1017-1028Crossref (132) 31Zhai Z. Yuan isolation characterization Pik, emerged after domestication.New 189: 321-334Crossref (150) 32Zhai Yao Liu Function coupled responsible Pik-h encoded resistance.PLoS One. 9e98067Crossref (56) located domain. resemble target AVR-Pik. it has speculated may (33Guo Cesari de Guillen Mammri Meusnier Bonnot Padilla Peng Y.-L. Specific two MAX involves distinct surfaces.Proc. Natl. Acad. U. 2018; 115: 11637Crossref (27) until recently (34Oikawa Shimizu Takeda Białas Kellner Bozkurt T.O. Maidment J.H.R. et al.The stabilizes heavy metal-associated co-opt immunity.bioRxiv. https://doi.org/10.1101/2020.12.01.406389Crossref (0) Scholar) remained undefined.Figure 2AVR-PikD affinity vitro. A, normalized traces AVR-PikD alone (green) (black). SDS-PAGE shows fractions peak elution volumes each sample, demonstrating coelute. absorbs UV light 280 nm very poorly (molar extinction coefficient 360 cm−1M−1), so no visible OsHIPP19-HMA. B, multicycle kinetics data resonance (colored lines) 1-to-1 fitted (black residuals plotted below. Green red acceptance thresholds determined Biacore T100 evaluation software.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3AVR-PikD higher Pikp-1 Pikm-1. schematic representation Pikm-1, OsHIPP19. shares 51% identity both Pikp-HMA Pikm-HMA. amino acid alignment OsHIPP19, Pikp-1, was produced Clustal Omega colored using BoxShade. C, boxplots showing %Rmax observed interactions percentage theoretical maximum response, assuming 1:1 HMA:effector Pikm-HMA, 2:1 PikpE230R-HMA. center line box represents median limits upper lower quartiles. whiskers smallest value within Q1 − 1.5× interquartile range (IQR) largest Q3 + IQR. Individual points represented black shapes. experiment repeated three times, consisting technical replicates. Plots were ggplot2 package (71Wickham ggplot2: Elegant Graphics Data Analysis. Springer-Verlag, New York2016Crossref (72R Core Development TeamR: Language Environment Statistical Computing. Foundation Computing, Vienna, Austria2018Google Scholar).View (PPT) Six (A–F) differing just five positions (28Kanzaki 35Yoshida Fujisawa Matsumura Takano Association genetics oryzae.Plant 21: 1573-1591Crossref (270) 36Wu Li Stepwise arms AvrPik pathosystem.Mol. 27: 759-769Crossref (22) 37Longya Chaipanya Jantasuriyarat Gene duplication mutation emergence aggressive allele fungus.Mol. 2019; 32: 740-749Crossref (14) Each polymorphic acids Pik-1-HMA indicating adaptive. carrying lines Pikp, Pikm, Pikh, Pik∗, AVR-PikE AVR-PikA elicit Pikm Pikh 38De la Concepcion J.C. Longya Xiao allelic extended single polymorphism interface.bioRxiv. https://doi.org/10.1101/2020.09.05.284240Crossref No known naturally occurring respond AVR-PikF. independent study yeast two-hybrid screen. Four small domain-containing (sHMA) interactors AVR-PikD. (LOC_Os04g39350) OsHIPP20 (LOC_Os04g39010) members (HIPP) family (39de Abreu-Neto J.B. Turchetto-Zolet A.C. Oliveira L.F.V. Bodanese Zanettini M.H. Margis-Pinheiro Heavy (HIPP): Characterization exclusive plants.FEBS 280: 1604-1616Crossref (89) 40Khan I.U. Rono J.K. B.Q. X.S. M.Q. L.L. X.C. Cao H.W. Yang Z.M. Identification (Oryza sativa) HPP HIPP tolerant metal toxicity.Ecotoxicol. Environ. Saf. 175: 8-18Crossref (37) while OsHPP03 (LOC_Os02g37290) OsHPP04 (LOC_Os02g37300) belong (HPP) family. All four contain CααX isoprenylation motif (-CSIM) C termini (Fig. 1). taking approach, characterize high tighter alleles. Further, tight AVR-PikD, but shared variants. Finally, present revealing effector-binding findings reveal protein’s putative virulence-associated opportunities target-guided IDs profile. study, Of these, selected further frequently initial screen Based well-characterized 41De MacLean Protein expands profile 8e47713Crossref (39) 42De Maqbool Polymorphic residues pathogen.Nat. Plants. 4: 576-585Crossref hypothesized would defined (OsHIPP19-HMA hereafter) 1 77 inclusive. This expressed purified Escherichia coli (see Experimental procedures) used subsequent Intact mass spectrometry confirmed 8323 Da exactly matched mass. First, qualitatively test When combined, coeluted column, earlier volume alone, form vitro 2A). Phytophthora infestans PexRD54, autophagy-related ATG8 its (Solanum tuberosum) (43Dagdas Y.F. Belhaj Chaparro-Garcia Pandey Petre Tabassum Cruz-Mireles Hughes R.K. Sklenar Menke Findlay al.An Irish potato famine antagonizes autophagy cargo 23Crossref (113) 44Maqbool Tregidgo Zess Round 8 (ATG8) PexRD54.J. Chem. 291: 20270-20282Abstract (45) negative control did coelute OsHIPP19-HMA, nor shift PexRD54 S1). To investigate (SPR). immobilized Ni2+-NTA chip via noncleavable 6xHis tag terminus protein. concentrations 0.25 nM 25 nM, flowed over chip. fit 2B) estimate rate constants ka kd (Table S1), equilibrium dissociation constant, KD, could calculated (KD = kd/ka). KD obtained shown 2B 0.7 nM. replicates performed similar 0.6 0.9 nM). conclude Pikm-1 3, B). again SPR. tag. Three (2 5 20 nM) chip, (Robs, measured units (RU)) recorded. Robs then (Rmax) if molecule bound referred %Rmax. Mutating glutamate-230 arginine predicted disrupt d