The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.

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The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

10.1101/2020.09.05.284240 ◽

2020 ◽

Cited By ~ 1

Author(s):

Juan Carlos De la Concepcion ◽

Josephine H. R. Maidment ◽

Apinya Longya ◽

Gui Xiao ◽

Marina Franceschetti ◽

...

Keyword(s):

Immune Responses ◽

Rice Blast ◽

Immune Recognition ◽

Immune Receptor ◽

Immune Receptors ◽

Pathogen Effectors ◽

Binding Interface ◽

Recognition Specificity ◽

Blast Fungus ◽

Effector Recognition

AbstractArms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.Author SummaryPlant pathogens constantly evolve to overcome immune defences and successfully colonize hosts, resulting in some of the most devastating diseases that affect global food production. To defend themselves, plants have evolved a sophisticated immune system that recognizes the presence of different pathogens and triggers immune responses to stop their spread. How plant immune receptors achieve extended recognition to specific pathogen strains and the molecular details of this recognition are just starting to be understood.In this study, we characterize how an allele of a rice immune receptor achieves a broad-spectrum recognition to effectors from the rice blast fungus. We found that this receptor has evolved a single change that alters the way it binds to different effector variants. This change increases binding affinity to these variants and this is ultimately translated to immune recognition. Interestingly, a different rice immune receptor allele also achieves broad-spectrum effector recognition in a similar way. Therefore, different immune receptor alleles can converge on a similar mechanism to achieve extended recognition to pathogen effectors.This knowledge has the potential to help to the rational design of plant immune receptors with bespoke resistance to some of the most destructive pathogens. A long-term goal in plant biotechnology.

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

eLife ◽

10.7554/elife.47713 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

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.

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

10.1101/611152 ◽

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.

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In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

Genetics ◽

10.1534/genetics.109.106013 ◽

2009 ◽

Vol 183 (3) ◽

pp. 853-860 ◽

Cited By ~ 31

Author(s):

Leila Feiz ◽

Brian S. Beecher ◽

John M. Martin ◽

Michael J. Giroux

Keyword(s):

Functional Analysis ◽

Allelic Variation ◽

Screening Method ◽

Point Mutations ◽

Missense Mutations ◽

Grain Hardness ◽

In Planta ◽

Rich Region ◽

Critical Function ◽

The Impact

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F2:F3 populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.

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Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk-disease complex

10.1101/099275 ◽

2017 ◽

Author(s):

Abraham Morales-Cruz ◽

Gabrielle Allenbeck ◽

Rosa Figueroa-Balderas ◽

Vanessa E. Ashworth ◽

Daniel P. Lawrence ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transcriptional Profiling ◽

Underlying Disease ◽

In Planta ◽

Dna And Rna ◽

Trunk Disease ◽

Specificity And Sensitivity ◽

Genome Wide ◽

Field Samples ◽

Trunk Diseases

SummaryGrapevines, like other perennial crops, are affected by so-called ‘trunk diseases’, which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e., metatranscriptomics) approach that can monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for mapping and quantifying DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally-infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.

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MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

10.1101/2020.07.08.194100 ◽

2020 ◽

Author(s):

Ryan Ard ◽

Jean-Christian Maillet ◽

Elias Daher ◽

Michael Phan ◽

Radoslav Zinoviev ◽

...

Keyword(s):

Catalytic Activity ◽

Molecular Mechanisms ◽

Rho Gtpase ◽

Pdz Domain ◽

Diacylglycerol Kinase ◽

Binding Motif ◽

Membrane Blebbing ◽

Rhoa Activity ◽

C Terminus ◽

Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

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Cellular functions and molecular mechanisms of non-lysine ubiquitination

Open Biology ◽

10.1098/rsob.190147 ◽

2019 ◽

Vol 9 (9) ◽

pp. 190147 ◽

Cited By ~ 18

Author(s):

Amie J. McClellan ◽

Sophie Heiden Laugesen ◽

Lars Ellgaard

Keyword(s):

Dna Repair ◽

Molecular Mechanisms ◽

Future Perspectives ◽

Cellular Functions ◽

The Past ◽

Protein Ubiquitination ◽

Open Questions ◽

Lysine Residues ◽

Molecular Machinery ◽

Mechanistic Basis

Protein ubiquitination is of great cellular importance through its central role in processes such as degradation, DNA repair, endocytosis and inflammation. Canonical ubiquitination takes place on lysine residues, but in the past 15 years non-lysine ubiquitination on serine, threonine and cysteine has been firmly established. With the emerging importance of non-lysine ubiquitination, it is crucial to identify the responsible molecular machinery and understand the mechanistic basis for non-lysine ubiquitination. Here, we first provide an overview of the literature that has documented non-lysine ubiquitination. Informed by these examples, we then discuss the molecular mechanisms and cellular implications of non-lysine ubiquitination, and conclude by outlining open questions and future perspectives in the field.

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Parthenogenesis as a Solution to Hybrid Sterility: The Mechanistic Basis of Meiotic Distortions in Clonal and Sterile Hybrids

Genetics ◽

10.1534/genetics.119.302988 ◽

2020 ◽

Vol 215 (4) ◽

pp. 975-987 ◽

Cited By ~ 1

Author(s):

Dmitrij Dedukh ◽

Zuzana Majtánová ◽

Anatolie Marta ◽

Martin Pšenička ◽

Jan Kotusz ◽

...

Keyword(s):

Gene Flow ◽

Molecular Mechanisms ◽

Hybrid Sterility ◽

Triploid Hybrid ◽

Interspecific Gene Flow ◽

Homologous Chromosomes ◽

Specific Manner ◽

Bivalent Formation ◽

Mechanistic Basis ◽

Sterile Hybrids

Hybrid sterility is a hallmark of speciation, but the underlying molecular mechanisms remain poorly understood. Here, we report that speciation may regularly proceed through a stage at which gene flow is completely interrupted, but hybrid sterility occurs only in male hybrids whereas female hybrids reproduce asexually. We analyzed gametogenic pathways in hybrids between the fish species Cobitis elongatoides and C. taenia, and revealed that male hybrids were sterile owing to extensive asynapsis and crossover reduction among heterospecific chromosomal pairs in their gametes, which was subsequently followed by apoptosis. We found that polyploidization allowed pairing between homologous chromosomes and therefore partially rescued the bivalent formation and crossover rates in triploid hybrid males. However, it was not sufficient to overcome sterility. In contrast, both diploid and triploid hybrid females exhibited premeiotic genome endoreplication, thereby ensuring proper bivalent formation between identical chromosomal copies. This endoreplication ultimately restored female fertility but it simultaneously resulted in the obligate production of clonal gametes, preventing any interspecific gene flow. In conclusion, we demonstrate that the emergence of asexuality can remedy hybrid sterility in a sex-specific manner and contributes to the speciation process.

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Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-16-0137-r ◽

2016 ◽

Vol 29 (8) ◽

pp. 651-660 ◽

Cited By ~ 17

Author(s):

Georgy Popov ◽

Malou Fraiture ◽

Frederic Brunner ◽

Guido Sessa

Keyword(s):

Pseudomonas Syringae ◽

Map Kinases ◽

Molecular Mechanisms ◽

Inducible Promoter ◽

Effector Proteins ◽

Host Cells ◽

In Planta ◽

Callose Deposition ◽

Type Iii ◽

Xanthomonas Euvesicatoria

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast–based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid–inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

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Structures of Phytophthora RXLR Effector Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m111.262303 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35834-35842 ◽

Cited By ~ 109

Author(s):

Laurence S. Boutemy ◽

Stuart R. F. King ◽

Joe Win ◽

Richard K. Hughes ◽

Thomas A. Clarke ◽

...

Keyword(s):

Immune System ◽

Molecular Mechanisms ◽

Tandem Repeats ◽

Effector Proteins ◽

Functional Adaptation ◽

In Planta ◽

Effector Domains ◽

The Core ◽

Plant Immune System ◽

Molecular Stability

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ∼44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.

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