Biology subjects

Maidment, J. H.

Publications and source records attributed to Maidment, J. H..

5 recordsLinked to original sources

bioRxiv2025

The Magnaporthe oryzae MAX effector AVR-Pia binds a novel group of rice HMA domain-containing proteins

Phytopathogenic fungi secrete effector proteins to promote virulence. MAX (Magnaporthe Avrs and ToxB-like) effectors form a sequence-diverse family sharing a conserved protein structure. AVR-Pia, a MAX effector from the rice blast fungus Magnaporthe oryzae, is recognised by the paired rice nucleotide-binding leucine-rich repeat immune receptors OsRGA4/OsRGA5 through direct binding to an integrated heavy metal-associated (HMA) domain in OsRGA5. Here, we identify previously unknown host targets of AVR-Pia: four HMA domain-containing rice proteins, belonging to the HMA Isoprenylated Plant Proteins (HIPPs) and HMA Plant Proteins (HPPs). AVR-Pia interacted with all four proteins, both in vitro and in planta, and bound their HMA domains with varying affinities. The crystal structure of AVR-Pia in complex with the HMA domain of OsHPP09 revealed the molecular details of the binding interface. Structure-guided mutagenesis of OsHPP09 identified a single point mutation which prevents AVR-Pia binding, providing a foundation for targeted engineering of HMA domains to evade effector binding.

plant biology↗

bioRxiv2023

pOPARA: Vectors for Golden Gate assembly of expression constructs containing the araBAD promoter

Heterologous protein production is often required to investigate the structural, biochemical, and biophysical properties of a protein of interest. Frequently, optimisation of expression conditions is required to obtain soluble protein and maximise yield. Trialling a variety of solubility and purification tags, as well as constructs containing different regulatory elements, is desirable. Golden Gate cloning allows modular assembly of different constructs using Type IIS restriction enzymes. The pOPIN vector suite, which utilises the T7 expression system, has been adapted to be compatible with Golden Gate assembly. Here, we present the pOPARA vectors. Expression from pOPARA vectors is driven by the araBAD promoter (pBAD) and is induced by addition of arabinose to the culture medium. pOPARA allows modular assembly of expression constructs using Golden Gate cloning with the CDS of interest and an optional C-terminal tag. pOPARA1 contains a carbenicillin resistance cassette flanked by restriction sites to allow exchange of the selectable markers. In pOPARA2, the carbenicillin resistance cassette has been exchanged for a spectinomycin resistance cassette. We demonstrate that both vectors can be used to express and produce a control protein.

molecular biology↗

bioRxiv2022

Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities.

Engineering expanded effector recognition in plant immune receptors is a promising prospect for generating new disease resistant crop varieties. However, modification of plant NLR receptors has proven challenging due to the lack of understanding of their context as part of complex immune systems. Here, we demonstrate a new avenue for NLR-mediated engineering that exploits the allelic diversity in the Pik NLR pair to allow for the generation of receptors with expanded recognition specificities, which would otherwise result in constitutive cell death. This work lays the foundation for the incorporation of new effector recognition motifs into the Pik system and advances the development of designer NLRs that can be tailored to specific secreted pathogen signatures. AbstractEngineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine rich repeat (NLRs) superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering novel disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. We have previously shown how alleles of the rice NLR pair Pik have differentially co-evolved, and how sensor/helper mismatching between non-co-evolved alleles triggers constitutive activation and cell death (De la Concepcion et al., 2021b). Here, we dissect incompatibility determinants in the Pik pair and found that HMA domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely co-evolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic HMA domains with novel and enhanced recognition of an effector from the rice blast fungus. Taken together, our results reveal a new strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

plant biology↗

bioRxiv2022

Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effectors host targets. Direct binding of effectors to these integrated domains activates plant defences. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defences. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants were resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to- nature disease resistance in crops. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/496076v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@23ec35org.highwire.dtl.DTLVardef@68b093org.highwire.dtl.DTLVardef@1209ab2org.highwire.dtl.DTLVardef@18c4adb_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

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. 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.

plant biology↗