Biology subjects

Franceschetti, M.

Publications and source records attributed to Franceschetti, M..

11 recordsLinked to original sources

bioRxiv2024

Iron and redox sensing in plants: properties of the hemerythrin-like domains of Arabidopsis BRUTUS and BRUTUS-LIKE2 proteins

Iron uptake in plants is negatively regulated by highly conserved hemerythrin (Hr) E3 ubiquitin ligases exemplified by Arabidopsis thaliana BRUTUS (BTS). Physiological studies suggest these are the elusive plant iron sensors, but biochemical evidence is lacking. Here we demonstrate that the N-terminal domains of BTS and BTS-LIKE2 (BTSL2) respectively bind three and two diiron centres within three closely packed Hr-like subdomains. The centres can be reversibly oxidized by O2 and H2O2, resulting in a di-Fe3+ form that is non-labile. In the reduced state, a proportion of the iron becomes labile, based on accessibility to Fe2+ chelators and reconstitution experiments, consistent with dynamic iron binding. Impaired iron binding and altered redox properties in the BTS dgl variant correlate with diminished capacity to suppress the downstream signalling cascade. These data provide the biochemical foundation for a mechanistic model of how BTS/Ls function as iron sensors that are unique to the plant kingdom.

biochemistry↗

bioRxiv2023

Genetic basis of historical pea mutants that hyper-accumulate iron

The Pisum sativum (pea) mutants degenerate leaves (dgl) and bronze (brz) accumulate large amounts of iron in leaves. First described several decades ago, the two mutants have provided important insights into iron homeostasis in plants but the underlying mutations have remained unknown. Using exome sequencing we identified an in-frame deletion associated with dgl in a BRUTUS homologue. The deletion is absent from wild type and the original parent line. BRUTUS belongs to a small family of E3 ubiquitin ligases acting as negative regulators of iron uptake in plants. The brz mutation was previously mapped to chromosome 4, and superimposing this region to the pea genome sequence uncovered a mutation in OPT3, encoding an oligopeptide transporter with a plant-specific role in metal transport. The causal nature of the mutations was confirmed by additional genetic analyses. Identification of the mutated genes rationalises many of the previously described phenotypes and provides new insights into shoot-to-root signalling of iron deficiency. Furthermore, the non-lethal mutations in these essential genes suggest new strategies for biofortification of crops with iron. Significance statementTwo iron-accumulating pea mutants first described more than 30 years ago have greatly contributed to our understanding of iron homeostasis in plants, but the mutations were never identified. Here we show that the phenotypes are caused by mutations in the BRUTUS and OPT3 genes and how this leads to specific defects in iron signalling and leaf development.

plant biology↗

bioRxiv2022

The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of the rice blast fungus

Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide binding site and leucine-rich repeat proteins. Like other Pik genes, both Piks-1 and Piks-2 are necessary and sufficient for Piks-mediated resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of M. oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. We conclude that Piks breaks the canonical Pik/AvrPik recognition pattern. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-1-HMA interact with AvrPik-A, -D and -E in vitro and in vivo, whereas Piks-1-HMA does not bind any AvrPik variants. Reciprocal exchanges of single amino acid residues between Piks-1 and Pikm-1 further reveal a dynamic recognition mechanism between Piks/Pikm alleles and their respective effectors. Piks-1E229Q/Pikm-1V261A can only activate immunity to AvrPik-D but not to other effectors, indicating that the amino acid change of E to Q at position 229 leads to its gain of a partial recognition spectrum of Pikm. By contrast, Piks-1A261V/Pikm-1Q229E confers immunity to the Piks cognate effector, indicating that the amino acid change of Q to E at position 229 leads to its shifts of the recognition from Pikm to Piks. Intriguingly, binding activities in both Y2H and analytical gel filtration assays are illustrated between Piks-1A261V/Pikm-1Q229E and AvrPik-D. However, it is unable to mount immunity against AvrPik-D, suggesting that biochemical activities based on in vitro and in vivo assays could be insufficient for sustaining biological function of receptor and effector pairs.

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↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗

bioRxiv2020

Multiple variants of the blast fungus effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19 with high affinity.

Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. Non-canonical integrated domains are widespread in plant NLRs and are thought to resemble the host target of the recognised effector. AVR-Pik interacts with specific rice HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.

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↗

bioRxiv2019

The Medicago truncatula Vacuolar Iron Transporter-Like proteins VTL4 and VTL8 deliver iron to endosymbiotic bacteria at different stages of the infection process

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron. The host plant is known to provide iron to developing bacteroids, but questions remain regarding which transporters are involved. Here, we characterize two Vacuolar Iron Transporter-Like (VTL) proteins in Medicago truncatula that are specifically expressed during nodule development. VTL4 is mostly expressed during early infection and the protein was localized to membranes and the infection thread. vtl4 mutants were delayed in nodule development. VTL8 is closely related to SEN1 in Lotus japonicus and expressed in the late stages of bacteroid differentiation. The VTL8 protein was localized to the symbiosome membrane. A mutant line lacking the tandemly-arranged VTL4 - VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a slight decrease in luminescence was observed in vtl4 mutants and a strong decrease in the 13U mutant. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. Taken together, these data indicate that VTL-type transporters are the main route for delivering iron to symbiotic rhizobia.

plant biology↗

bioRxiv2019

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

Plant 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.

plant biology↗

bioRxiv2019

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

Unconventional 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.

plant biology↗

bioRxiv2017

Lessons in effector and NLR biology of plant-microbe systems

A diversity of plant-associated organisms secrete effectors--proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

plant biology↗