scholarly journals Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistance

2019 ◽  
Author(s):  
Freya A. Varden ◽  
Hiromasa Saitoh ◽  
Kae Yoshino ◽  
Marina Franceschetti ◽  
Sophien Kamoun ◽  
...  
Keyword(s):  
Immune Response ◽  
Disease Resistance ◽  
Effector Proteins ◽  
In Planta ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains

ABSTRACTUnconventional integrated domains in plant intracellular immune receptors (NLRs) can directly bind translocated pathogen effector proteins to initiate an immune response. The rice immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the Magnaporthe oryzae effectors AVR-Pik and AVR-Pia, respectively. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains maintain specificity of recognition, even for structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance towards the “mismatched” effector AVR-Pia in planta, and that the Pikp-HMA domain binds AVR-Pia in vitro. The HMA domain from another Pik-1 allele, Pikm, is unable to bind AVR-Pia, and does not trigger a response in plants. The crystal structure of Pikp-HMA bound to AVR-Pia reveals a different binding interface compared to AVR-Pik effectors, suggesting plasticity in integrated domain/effector interactions. This work shows how a single NLR can bait multiple pathogen effectors via an integrated domain, and may enable engineering immune receptors with extended disease resistance profiles.

scholarly journals Protein engineering expands the effector recognition profile of a rice NLR immune receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Juan Carlos De la Concepcion ◽  
Marina Franceschetti ◽  
Dan MacLean ◽  
Ryohei Terauchi ◽  
Sophien Kamoun ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Direct Interaction ◽  
Structural Basis ◽  
In Planta ◽  
Pathogen Effectors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

scholarly journals Protein engineering expands the effector recognition profile of a rice NLR immune receptor

2019 ◽  
Author(s):  
JC De la Concepcion ◽  
M Franceschetti ◽  
R Terauchi ◽  
S Kamoun ◽  
MJ Banfield
Keyword(s):  
Protein Engineering ◽  
Direct Interaction ◽  
In Planta ◽  
Pathogen Effectors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains ◽  
Effector Recognition

AbstractPlant NLR receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this has proven challenging, in part due to their narrow response specificity. Here, we used structure-guided engineering to expand the response profile of the rice NLR Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector binding interface of the integrated Pikp-HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell death response to AVR-Pik variants previously unrecognized by Pikp in planta. Structures of the engineered Pikp-HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly via integrated domains.

scholarly journals Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25/26 E3 ligases for enhancing Verticillium wilt resistance in Arabidopsis

2021 ◽  
Author(s):  
Zhizhong Gong ◽  
Junsheng Qi ◽  
Aifang Ma ◽  
Dingpeng Zhang ◽  
Guangxing Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Plant Resistance ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Effector Proteins ◽  
In Planta ◽  
E3 Ligases

Verticillium wilt is a severe plant disease, increasing the plant resistance to this disease is a critical challenge worldwide. Here, we report that the Verticillium dahliae (V. dahliae)-secreted Aspf2-like protein VDAL causes leaf wilting when applied to cotton leaves in vitro, but enhances the resistance to V. dahliae when overexpressed in Arabidopsis or cotton. VDAL interacts with Arabidopsis E3 ligases PUB25 and PUB26 (PUBs) and is ubiquitinated by PUBs in vitro. However, VDAL is not degraded by PUBs in planta. Besides, the pub25 pub26 shows higher resistance to V. dahliae than the wild type. PUBs interact with the transcription factor MYB6 in a yeast two-hybrid screen. MYB6 promotes plant resistance to Verticillium wilt while PUBs ubiquitinate MYB6 and mediate its degradation. VDAL competes with MYB6 for binding to PUBs, and the role of VDAL in increasing wilt disease depends on MYB6. These results suggest that plants evolute a strategy to utilize the invaded effector protein VDAL to resist the V. dahliae infection without causing a hypersensitive response. This study provides the molecular mechanism for plants increasing disease resistance when overexpressing some effector proteins, and may promote searching for more genes from pathogenic fungi or bacteria to engineer plant disease resistance.

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

2021 ◽  
Author(s):  
Nitika Mukhi ◽  
Hannah Brown ◽  
Danylo Gorenkin ◽  
Pingtao Ding ◽  
Adam R Bentham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Immune Responses ◽  
Pseudomonas Syringae ◽  
Effector Proteins ◽  
Dna Binding Domain ◽  
Binding Affinities ◽  
Wrky Domain ◽  
In Planta ◽  
Integrated Domains

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

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

2021 ◽  
Vol 118 (50) ◽  
pp. e2113996118
Author(s):  
Nitika Mukhi ◽  
Hannah Brown ◽  
Danylo Gorenkin ◽  
Pingtao Ding ◽  
Adam R. Bentham ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Binding Domain ◽  
Effector Proteins ◽  
Binding Affinities ◽  
Wrky Domain ◽  
Nucleotide Binding Domain ◽  
In Planta ◽  
Integrated Domains ◽  
Effector Binding

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

scholarly journals An atypical N-ethylmaleimide sensitive factor enables the viability of nematode-resistant Rhg1 soybeans

2018 ◽  
Vol 115 (19) ◽  
pp. E4512-E4521 ◽  
Author(s):  
Adam M. Bayless ◽  
Ryan W. Zapotocny ◽  
Derrick J. Grunwald ◽  
Kaela K. Amundson ◽  
Brian W. Diers ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Soybean Cyst Nematode ◽  
In Planta ◽  
Attachment Protein ◽  
Binding Interface ◽  
Vitro Binding ◽  
Soybean Pest ◽  
Sensitive Factor ◽  
Resistance Trait

N-ethylmaleimide sensitive factor (NSF) and α-soluble NSF attachment protein (α-SNAP) are essential eukaryotic housekeeping proteins that cooperatively function to sustain vesicular trafficking. The “resistance to Heterodera glycines 1” (Rhg1) locus of soybean (Glycine max) confers resistance to soybean cyst nematode, a highly damaging soybean pest. Rhg1 loci encode repeat copies of atypical α-SNAP proteins that are defective in promoting NSF function and are cytotoxic in certain contexts. Here, we discovered an unusual NSF allele (Rhg1-associated NSF on chromosome 07; NSFRAN07) in Rhg1+ germplasm. NSFRAN07 protein modeling to mammalian NSF/α-SNAP complex structures indicated that at least three of the five NSFRAN07 polymorphisms reside adjacent to the α-SNAP binding interface. NSFRAN07 exhibited stronger in vitro binding with Rhg1 resistance-type α-SNAPs. NSFRAN07 coexpression in planta was more protective against Rhg1 α-SNAP cytotoxicity, relative to WT NSFCh07. Investigation of a previously reported segregation distortion between chromosome 18 Rhg1 and a chromosome 07 interval now known to contain the Glyma.07G195900 NSF gene revealed 100% coinheritance of the NSFRAN07 allele with disease resistance Rhg1 alleles, across 855 soybean accessions and in all examined Rhg1+ progeny from biparental crosses. Additionally, we show that some Rhg1-mediated resistance is associated with depletion of WT α-SNAP abundance via selective loss of WT α-SNAP loci. Hence atypical coevolution of the soybean SNARE-recycling machinery has balanced the acquisition of an otherwise disruptive housekeeping protein, enabling a valuable disease resistance trait. Our findings further indicate that successful engineering of Rhg1-related resistance in plants will require a compatible NSF partner for the resistance-conferring α-SNAP.

scholarly journals Identification of a putative DNA-binding protein in Arabidopsis that acts as a susceptibility hub and interacts with multiple Pseudomonas syringae effectors

2020 ◽  
Author(s):  
Karl Schreiber ◽  
Jennifer D Lewis
Keyword(s):  
Pseudomonas Syringae ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Ribosomal Protein Genes ◽  
Loss Of Function ◽  
Wild Type ◽  
In Planta ◽  
Arabidopsis Protein ◽  
Wide Range

Phytopathogens use secreted effector proteins to suppress host immunity and promote pathogen virulence, and there is increasing evidence that the host-pathogen interactome comprises a complex network. In an effort to identify novel interactors of the Pseudomonas syringae effector HopZ1a, we performed a yeast two-hybrid screen that identified a previously uncharacterized Arabidopsis protein that we designate HopZ1a Interactor 1 (ZIN1). Additional analyses in yeast and in planta revealed that ZIN1 also interacts with several other P. syringae effectors. We show that an Arabidopsis loss-of-function zin1 mutant is less susceptible to infection by certain strains of P. syringae, while overexpression of ZIN1 results in enhanced susceptibility. Functionally, ZIN1 exhibits topoisomerase-like activity in vitro. Transcriptional profiling of wild-type and zin1 Arabidopsis plants inoculated with P. syringae indicated that while ZIN1 regulates a wide range of pathogen-responsive biological processes, the list of genes more highly expressed in zin1 versus wild-type plants was particularly enriched for ribosomal protein genes. Altogether, these data illuminate ZIN1 as a potential susceptibility hub that interacts with multiple effectors to influence the outcome of plant-microbe interactions.

scholarly journals Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
A Maqbool ◽  
H Saitoh ◽  
M Franceschetti ◽  
CEM Stevenson ◽  
A Uemura ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Rice Blast ◽  
Plant Pathogens ◽  
Rice Cultivar ◽  
Blast Disease ◽  
Structural Basis ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Direct Recognition

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

scholarly journals An XA21-Associated Kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors

2013 ◽  
Author(s):  
Xuewei Chen ◽  
Shimin Zuo ◽  
Benjamin Schwessinger ◽  
Mawsheng Chern ◽  
Patrick Canlas ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Yeast Two Hybrid System ◽  
Intracellular Domains ◽  
Activity Dependent

The rice XA21 immune receptor kinase and the structurally related XA3 receptor, confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast-two-hybrid system in a kinase activity dependent manner. OsSERK2 undergoes bidirectional trans-phosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. Taken together, these results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function.

scholarly journals Unique Secreted in Xylem Genes in Banana-Infecting Endophytic Fusarium Oxysporum

Proceedings ◽  
2020 ◽  
Vol 36 (1) ◽  
pp. 180
Author(s):  
Rebecca Lyons ◽  
Elizabeth Czislowski ◽  
Isabel Zeil-Rolfe ◽  
Shubhdeep Kaur ◽  
Zhendong Liu ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Effector Proteins ◽  
In Planta ◽  
Banana Industry ◽  
Fusarium Wilt Of Banana ◽  
Race 4 ◽  
Fusarium Oxysporum Species Complex ◽  
Six Genes

Members of the Fusarium oxysporum species complex include pathogenic and non-pathogenic isolates and infect a broad range of plant species. F. oxysporum f. sp. cubense (Foc) causes the destructive Fusarium wilt of banana, and the recently emerged Foc tropical race 4 strain threatens the global banana industry. Secreted in xylem (SIX) genes encode for F. oxysporum effector proteins that are associated with virulence in pathogenic F. oxysporum, however they have rarely been reported from non-pathogenic F. oxysporum isolates. Our recent survey of asymptomatic banana plants grown in Foc-infested fields in Queensland and northern NSW revealed that diverse Fusarium spp, including F. oxysporum, reside in the plant roots and pseudostem without causing obvious damage to the plant. Intriguingly, we amplified SIX genes from several of the putative endophytic F. oxysporum isolates identified in the survey and found that they differ in their profile to known Foc SIX genes. To study the role of the endophytic F. oxysporum isolates in planta and the biological function of their SIX genes in more detail, we will re-inoculate cultivated and wild diploid banana lines with the endophytic F. oxysporum strains under glasshouse conditions to assess if they are non-pathogenic on banana. Secondly, we will determine whether the endophytic F. oxysporum SIX genes are expressed in planta and/or in vitro and look at the transcriptome changes occurring in the host following infection. Finally, endophytic F. oxysporum strains transformed with GFP will be used to investigate the extent of fungal colonisation in the plant.

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