Biology subjects

Kroj, T.

Publications and source records attributed to Kroj, T..

9 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↗

bioRxiv2024

Zymoseptoria tritici proteins structurally related to UmV-KP4 and UmV-KP6 are toxic to fungi, and define novel structural families of fungal effectors

Fungal effectors play crucial roles in plant infection. Despite low sequence identity, effectors were recently classified into structural families. In this study, we have elucidated the structures of Zt-NIP1 and Mycgr3-91409 effectors of the wheat fungal pathogen Zymoseptoria tritici, using X-ray crystallography and NMR. These effectors displayed a structural homology with, respectively, KP4 and KP6 killer toxins, from UmV dsRNA viruses infecting the corn fungal pathogen Ustilago maydis. Consequently, Zt-NIP1 and Mycgr3-91409 were renamed Zt-KP4-1 and Zt-KP6-1. Orthologs and paralogs of Zt-KP4-1 and Zt-KP6-1 were identified in Zymoseptoria, but not in other fungi, except Ecp2 effectors related to Zt-KP4-1. Assessment of the biological activities of Zt-KP6-1 and Zt-KP4-1 revealed their toxicity to fungi such as Botrytis cinerea and Z. tritici, but not to wheat. A novel pipeline relying on Foldseek and cysteine-pattern constrained HMM searches of AlphaFold2 predicted structures from Uniprot generated a comprehensive inventory of KP4 and KP6 proteins in fungi and plants. A structure-based classification of these proteins revealed four KP4 and three KP6 structural super families. This classification provided far-reaching hypotheses on their biological function and evolution. This unifying structural framework highlights the power of structure determination for the classification of effectors, and their functional investigation.

biochemistry↗

bioRxiv2024

Rice JASMONIC ACID OXIDASES (OsJAO) control resting jasmonate metabolism to promote development and repress basal immune responses

Recent research has established that catabolic conversions within the jasmonate pathway have significant consequences on hormone signaling output. In dicotyledonous plants, the jasmonic acid oxidase (JAO) catabolic route is endowed with a regulatory function by diverting jasmonic acid (JA) towards hydroxylation, at the expense of its conjugation into the bioactive jasmonoyl-isoleucine (JA-Ile) hormone. Here we functionally characterized the JAO pathway in rice (Oryza sativa) and demonstrate its prevalent function in promoting growth and attenuating JA responses in vegetative tissues. The rice genome contains four JAO-related homologs of which three generated hydroxy-JA in vitro and reverted the high defense phenotype when expressed in the Arabidopsis jao2-2 mutant. By generating and analyzing a series of single to quadruple rice jao mutants, we show the incremental effect of gradual JAO depletion on JA metabolism, basal defense levels, growth inhibition, fitness and global metabolic reprogramming. JAO-deficient lines were significantly growth-retarded at the juvenile stage, while recovering a near wild-type vegetative development after three months, where they exhibited a enhanced resistance to virulent and avirulent strains of Magnaporthe oryzae, the causal agent of fungal blast disease. Our findings identify the JAO pathway as an integral component of rice JA homeostasis and an important determinant of the growth-defense tradeoff. They demonstrate its conserved regulatory function in monocots and open possibilities for modulating selectively basal JA responses in a major cereal crop. Natural variation in JAO activity could also be explored as a mechanism underlying varying levels of JA signaling output in rice.

plant biology↗

bioRxiv2024

Immune regulators EDS1 and PAD4 constrain ADR1-dependent disease resistance in rice

Withdrawal StatementThe authors have withdrawn this manuscript owing to finding inconsistencies in genotyping of kitaake rice eds1 pad4 double mutant lines and to establishing that combined loss of EDS1 and PAD4 does not underlie the stunted autoimmune-like phenotype reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors: Jane Parker, Thomas Kroj and Haitao Cui (co-corresponding)

plant biology↗

bioRxiv2023

The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a {beta}-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets. Author summaryMAX effectors are a family of virulence proteins from the plant pathogenic fungus Pyricularia (syn. Magnaporthe) oryzae that share a similar 3D structure despite very low amino-acid sequence identity. Characterizing the function and evolution of these proteins requires a detailed understanding of their structural diversity. With this in mind, we have determined the NMR structures of four new MAX effectors and shown a near-perfect match with the corresponding AlphaFold2 (AF2) models. We then applied a prediction pipeline based on similarity searches with structural modeling using the AF2 software to predict MAX effectors in a collection of 120 P. oryzae genomes. The resulting models and experimental structures revealed that the MAX core while preserved is highly permissive to secondary structure variations and may coexists with extensive structural diversity in terms of structured N- or C-terminal extensions permitting their classification. For a subset of AF2 models, we have also analyzed the physico-chemical properties of the core domain surfaces, adding another, more functional perspective, notably surface electrostatics and stickiness. This work constitutes a major step in understanding the relationships among MAX effectors by analyzing their structural landscape and cataloguing specific physico-chemical properties. It also provides valuable insights for guiding research into the putative targets of these effectors in infected plant hosts.

bioinformatics↗

bioRxiv2023

Adaptive evolution in virulence effectors of the rice blast fungus Pyricularia oryzae

Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae. Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors. AUTHOR SUMMARYFungal plant pathogens use small secreted proteins, called effectors, to manipulate to their own advantage their hosts physiology and immunity. The evolution of these effectors, whether spontaneously or in response to human actions, can lead to epidemics or the emergence of new diseases. It is therefore crucial to understand the mechanisms underlying this evolution. In this article, we report on the evolution of effectors in one of the prime experimental model systems of plant pathology, Pyricularia oryzae, the fungus causing blast diseases in rice, wheat, and other cereals or grasses. We further characterize in this fungus a particular class of effectors, called MAX effectors, using structural models based on experimental protein structures of effectors. We show that this class of effector is produced by the pathogen during the early stages of infection, when plant cells are still alive. By comparing the gene content of isolates infecting different plant species, we show that the MAX effector arsenal is highly variable from one isolate to another. Finally, using the inferential framework of population genetics, we demonstrate that MAX effectors exhibit very high genetic variability and that this results from the action of natural selection.

evolutionary biology↗

bioRxiv2022

Extensive immune receptor repertoire diversity in disease-resistant rice landraces

Plants have powerful defence mechanisms, and extensive immune receptor repertoires, yet crop monocultures are prone to epidemic diseases. Rice is susceptible to many diseases, such as rice blast caused by Magnaporthe oryzae. Varietal resistance of rice to blast relies on intracellular nucleotide binding, leucine-rich repeat (NLR) receptors that recognize specific pathogen molecules and trigger immune responses. In the Yuanyang terraces in south-west China, rice landraces rarely show severe losses to disease whereas commercial inbred lines show pronounced field susceptibility. Here, we investigate within-landrace NLR sequence diversity of nine rice landraces and eleven modern varieties of indica, japonica and aus using complexity reduction techniques. We find that NLRs display high sequence diversity in landraces, consistent with balancing selection, and that balancing selection at NLRs is more pervasive in landraces than modern varieties. Notably, modern varieties lack many ancient NLR haplotypes that are retained in some landraces. Our study emphasises the value of standing genetic variation that is maintained in farmer landraces as resource to make modern crops and agroecosystems less prone to disease.

evolutionary biology↗

bioRxiv2021

Design of a new effector recognition specificity in a plant NLR immune receptor by molecular engineering of its integrated decoy domain

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that specifically recognize pathogen effectors and induce immune responses. Designing artificial NLRs with new effector recognition specificities is a promising prospect for sustainable, knowledge-driven crop protection. However, such strategies are hampered by the complexity of NLR function. Here, we tested whether molecular engineering of the integrated decoy domain (ID) of an NLR could extend its recognition spectrum to a new effector. To this aim, we relied on the detailed molecular knowledge of the recognition of distinct Magnaporthe oryzae MAX (Magnaporthe AVRs and ToxB-like) effectors by the rice NLRs RGA5 and Pikp-1. For both NLRs, effector recognition involves physical binding to their HMA (Heavy Metal-Associated) IDs. However, AVR-PikD, the effector recognized by Pikp-1, binds to a completely different surface of the HMA domain compared to AVR-Pia and AVR1-CO39, recognized by RGA5. By introducing into the HMA domain of RGA5 the residues of the Pikp-1 HMA domain involved in AVR-PikD binding, we created a high-affinity binding surface for this new effector. In the Nicotiana benthamiana heterologous system, RGA5 variants carrying this engineered binding surface still recognize AVR-Pia and AVR1-CO39, but also perceive the new ligand, AVR-PikD, resulting in the activation of immune responses. Therefore, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs. However, it pinpoints significant knowledge gaps that limit the full deployment of this NLR-ID engineering strategy and provides hypotheses for future research on this topic.

plant biology↗

bioRxiv2020

The blast pathogen effector AVR-Pik binds and stabilizes rice heavy metal-associated (HMA) proteins to co-opt their function in immunity

Intracellular nucleotide-binding domain and leucine-rich repeat-containing (NLR) receptors play crucial roles in immunity across multiple domains of life. In plants, a subset of NLRs contain noncanonical integrated domains that are thought to have evolved from host targets of pathogen effectors to serve as pathogen baits. However, the functions of host proteins with similarity to NLR integrated domains and the extent to which they are targeted by pathogen effectors remain largely unknown. Here, we show that the blast fungus effector AVR-Pik binds a subset of related rice proteins containing a heavy metal-associated (HMA) domain, one of the domains that has repeatedly integrated into plant NLR immune receptors. We find that AVR-Pik binding stabilizes the rice small HMA (sHMA) proteins OsHIPP19 and OsHIPP20. Knockout of OsHIPP20 causes enhanced disease resistance towards the blast pathogen, indicating that OsHIPP20 is a susceptibility gene (S-gene). We propose that AVR-Pik has evolved to bind HMA domain proteins and co-opt their function to suppress immunity. Yet this binding carries a trade-off, it triggers immunity in plants carrying NLR receptors with integrated HMA domains. Significance StatementRice blast disease, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. Therefore, understanding the mechanisms of blast fungus infection and resistance of rice against the disease is important for global food security. In this study, we show that the M. oryzae effector protein AVR-PikD binds rice sHMA proteins and stabilizes them, presumably to enhance pathogen infection. We show that loss-of-function mutants in one rice sHMA, OsHIPP20, reduced the level of susceptibility against a compatible isolate of M. oryzae, suggesting that M. oryzae requires host sHMA to facilitate invasion. Remarkably, OsHIPP20 knockout rice line showed no growth defect, suggesting editing sHMA genes may present a novel source of resistance against blast disease.

plant biology↗