Heavy Metal-associated Domain Research Articles

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 effector's host targets. Direct binding of effectors to these integrated domains activates plant defenses. 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 defenses. 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 the 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 was 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.

Genome-wide survey of HMA gene family and its characterization in wheat (Triticum aestivum).

Abiotic stresses, particularly drought and heavy metal toxicity, have presented a significant risk to long-term agricultural output around the world. Although the heavy-metal-associated domain (HMA) gene family has been widely explored in Arabidopsis and other plants, it has not been thoroughly studied in wheat (Triticum aestivum). This study was proposed to investigate the HMA gene family in wheat. To analyze the phylogenetic relationships, gene structure, gene ontology, and conserved motifs, a comparative study of wheat HMA genes with the Arabidopsis genome was performed. A total of 27 T. aestivum proteins belonging to the HMA gene family were identified in this study, with amino acid counts ranging from 262 to 1,071. HMA proteins were found to be grouped into three subgroups in a phylogenetic tree, and closely related proteins in the tree showed the same expression patterns as motifs found in distinct subgroups. Gene structural study elucidated that intron and exon arrangement differed by family. As a result, the current work offered important information regarding HMA family genes in the T. aestivum genome, which will be valuable in understanding their putative functions in other wheat species.

Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains

BackgroundA copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCSdI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCSdI in CCS. MethodsTo evaluate protein-protein interactions of CCS through CCSdI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed. ResultsWe found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCSdI. The interaction between CCSdI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCSdI and HAH1. ConclusionWhile physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCSdI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.

The Barley Heavy Metal Associated Isoprenylated Plant Protein HvFP1 Is Involved in a Crosstalk between the Leaf Development and Abscisic Acid-Related Drought Stress Responses.

The heavy metal associated isoprenylated plant proteins (HIPPs) are characterized by at least one heavy metal associated (HMA) domain and a C-terminal isoprenylation motif. Hordeum vulgare farnesylated protein 1 (HvFP1), a barley HIPP, is upregulated during drought stress, in response to abscisic acid (ABA) and during leaf senescence. To investigate the role of HvFP1, two independent gain-of-function lines were generated. In a physiological level, the overexpression of HvFP1 results in the delay of normal leaf senescence, but not in the delay of rapid, drought-induced leaf senescence. In addition, the overexpression of HvFP1 suppresses the induction of the ABA-related genes during drought and senescence, e.g., HvNCED, HvS40, HvDhn1. Even though HvFP1 is induced during drought, senescence and the ABA treatment, its overexpression suppresses the ABA regulated genes. This indicates that HvFP1 is acting in a negative feedback loop connected to the ABA signaling. The genome-wide transcriptomic analysis via RNA sequencing revealed that the gain-of-function of HvFP1 positively alters the expression of the genes related to leaf development, photomorphogenesis, photosynthesis and chlorophyll biosynthesis. Interestingly, many of those genes encode proteins with zinc binding domains, implying that HvFP1 may act as zinc supplier via its HMA domain. The results show that HvFP1 is involved in a crosstalk between stress responses and growth control pathways.

The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a

BackgroundCadmium (Cd2+) is one of the highly toxic heavy metals and is considered as a carcinogenic agent. Our aim was to confirm the ability of Frankia alni ACN14a to resist Cd2+ and to determine the genes involved in the resistance mechanism. ResultsF. alni ACN14a and Frankia casuarinae CcI3 hyphae showed up to 10 and 22 times Cd2+ accumulation when exposed to 1 mM Cd2+, respectively. Scanning electron microscopy (SEM) exhibited a stable Cd2+ precipitate on the cell surface, and the increase in Cd2+ weight level reached 16.45% when evaluated with SEM-EDAX analysis. The following two potential Cd2+ operons were identified: 1. cadCA operon, which encodes a copper-transporting P-type ATPase A (cadA, FRAAL0989) and an ArsR family regulator (cadC, FRAAL0988), with 37- and 70-fold increase in their expression by qRT-PCR, respectively and 2. cadB/DX, which encodes a putative cobalt-zinc-cadmium resistance protein (cadD, FRAAL3628) and heavy metal-associated domain protein (cadX, FRAAL3626), with 22- and 16-fold upregulation when exposed to Cd2+ stress. ConclusionsCd2+ tolerance by F. alni ACN14a involved efflux of Cd2+ outside the cells and binding it to the membrane surface. Our results indicate the existence of two cadmium-resistance mechanisms in Frankia strains, which support the idea of using them as a bioremediation agent.How to cite: Rehan M, Alhusays A, Serag AM, et al. The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a. Electron J Biotechnol 2022;60. https://doi.org/10.1016/j.ejbt.2022.09.006.

The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction.

The rice nucleotide‐binding (NB) and leucine‐rich repeat (LRR) domain immune receptors (NLRs) RGA4 and RGA5 form a helper NLR/sensor NLR (hNLR/sNLR) pair that specifically recognizes the effectors AVR‐Pia and AVR1‐CO39 from the blast fungus Magnaporthe oryzae. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal‐associated (HMA) domain integrated after its LRR domain. This RGA5HMA domain binds the effectors and is crucial for their recognition. Investigation of the three‐dimensional structure of the AVR1‐CO39/RGA5HMA complex by X‐ray crystallography identified a candidate surface for effector binding in the HMA domain and showed that the HMA domain self‐interacts in the absence of effector through the same surface. Here, we investigated the relevance of this HMA homodimerization for RGA5 function and the role of the RGA5HMA effector‐binding and self‐interaction surface in effector recognition. By analysing structure‐informed point mutations in the RGA5HMA‐binding surface in protein interaction studies and in Nicotiana benthamiana cell death assays, we found that HMA self‐interaction does not contribute to RGA5 function. However, the effector‐binding surface of RGA5HMA identified by X‐ray crystallography is crucial for both in vitro and in vivo effector binding as well as effector recognition. These results support the current hypothesis that noncanonical integrated domains of NLRs act primarily as effector traps and deepen our understanding of the sNLRs' function within NLR pairs.

EMS-based mutants are useful for enhancing drought tolerance in spring wheat

Sustainable wheat production in drought prone areas can be achieved by developing resilient wheat varieties. In the present study, chemical mutagenesis was used to induce mutations in a cultivated wheat variety ‘NN-Gandum-1’. In total, 44 mutants were selected based on their high yield potential for exposing to well-watered (W 1) and rainfed (W 2) conditions for one season. Then, 24 mutants were selected and were exposed to W 1 and W 2 regimes. On the basis of least relative reduction in physiological parameters under W 2 regime, five mutants were selected for conducting exome capturing assays. In total, 184 SNPs were identified in nine genes (ABC transporter type 1, aspartic peptidase, cytochrome P450, transmembrane domain, heavy metal-associated domain, HMA, NAC domain, NAD (P)-binding domain, S-type anion channel, Ubiquitin-conjugating enzyme E2 and UDP-glucuronosyl/UDP-glucosyltransferase). Maximum number of mutations were observed in chr.2D, which contained mutations in three genes, i.e. ABC transporter type 1, NAD (P)-binding domain and UDP-glucuronosyl/UDP-glucosyltransferase which may have a role in conferring drought tolerance. The selected mutants were further tested for studying their biochemical responses under both the regimes for 2 years. The extent of membrane damage was estimated through malondialdehyde and hydrogen per oxidase, and tolerance to drought stress was assessed via antioxidant enzymes in leaves. The selected mutants under drought stress increased the accumulation of proline content, total soluble sugars, total free amino acids, while decreased total chlorophyll content, carotenoids and total soluble protein. These mutants can further be explored to understand the genetic circuits of drought tolerance in wheat.

Genome-wide identification and function analysis of HMAD gene family in cotton (Gossypium spp.)

BackgroundThe abiotic stress such as soil salinization and heavy metal toxicity has posed a major threat to sustainable crop production worldwide. Previous studies revealed that halophytes were supposed to tolerate other stress including heavy metal toxicity. Though HMAD (heavy-metal-associated domain) was reported to play various important functions in Arabidopsis, little is known in Gossypium.ResultsA total of 169 G. hirsutum genes were identified belonging to the HMAD gene family with the number of amino acids ranged from 56 to 1011. Additionally, 84, 76 and 159 HMAD genes were identified in each G. arboreum, G. raimondii and G. barbadense, respectively. The phylogenetic tree analysis showed that the HMAD gene family were divided into five classes, and 87 orthologs of HMAD genes were identified in four Gossypium species, such as genes Gh_D08G1950 and Gh_A08G2387 of G. hirsutum are orthologs of the Gorai.004G210800.1 and Cotton_A_25987 gene in G. raimondii and G. arboreum, respectively. In addition, 15 genes were lost during evolution. Furthermore, conserved sequence analysis found the conserved catalytic center containing an anion binding (CXXC) box. The HMAD gene family showed a differential expression levels among different tissues and developmental stages in G. hirsutum with the different cis-elements for abiotic stress.ConclusionsCurrent study provided important information about HMAD family genes under salt-stress in Gossypium genome, which would be useful to understand its putative functions in different species of cotton.

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain.

A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

Molecular mechanisms of heavy metals resistance of Stenotrophomonas rhizophila JC1 by whole genome sequencing.

In this study, a higher metal ions-resistant bacterium, Stenotrophomonas rhizophila JC1 was isolated from contaminated soil in Jinchang city, Gansu Province, China. The Pb2+ (120mg/L) and Cu2+ (80mg/L) removal rate of the strain reached at 76.9% and 83.4%, respectively. The genome comprises 4268161bp in a circular chromosome with 67.52% G + C content and encodes 3719 proteins. The genome function analysis showed czc operon, mer operon, cop operon, arsenic detoxification system in strain JC1 were contributed to the removal of heavy metals. Three efflux systems (i.e., RND, CDF, and P-ATPase) on strain JC1 genome could trigger the removal of divalent cations from cells. cAMP pathway and ABC transporter pathway might be involved in the transport and metabolism of heavy metals. The homology analysis exhibited multi-gene families such as ABC transporters, heavy metal-associated domain, copper resistance protein, carbohydrate-binding domain were distributed across 410 orthologous groups. In addition, heavy metal-responsive transcription regulator, thioredoxin, heavy metal transport/detoxification protein, divalent-cation resistance protein CutA, arsenate reductase also played important roles in the heavy metals adsorption and detoxification process. The complete genome data provides insight into the exploration of the interaction mechanism between microorganisms and heavy metals.

Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense

Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized 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 recognized by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a noncanonical integrated heavy-metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defenses. The host targets of AVR-Pik are also HMA-domain-containing proteins, namely heavy-metal-associated isoprenylated plant proteins (HIPPs) and heavy-metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared with the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19 and compare the interaction to that formed 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 characterized 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 unrecognized AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.

Heavy Metal Transporters-Associated Proteins in S. tuberosum: Genome-Wide Identification, Comprehensive Gene Feature, Evolution and Expression Analysis.

Plants have evolved a number of defense and adaptation responses to protect themselves against challenging environmental stresses. Genes containing a heavy metal associated (HMA) domain are required for the spatiotemporal transportation of metal ions that bind with various enzymes and co-factors within the cell. To uncover the underlying mechanisms mediated by StHMA genes, we identified 36 gene members in the StHMA family and divided them into six subfamilies by phylogenetic analysis. The StHMAs had high collinearity and were segmentally duplicated. Structurally, most StHMAs had one HMA domain, StHIPPc and StRNA1 subfamilies had two, and 13 StHMAs may be genetically variable. The StHMA gene structures and motifs varied considerably among the various classifications, this suggests the StHMA family is diverse in genetic functions. The promoter analysis showed that the StHMAs had six main cis-acting elements with abiotic stress. An expression pattern analysis revealed that the StHMAs were expressed tissue specifically, and a variety of abiotic stresses may induce the expression of StHMA family genes. The HMA transporter family may be regulated and expressed by a series of complex signal networks under abiotic stress. The results of this study may help to establish a theoretical foundation for further research investigating the functions of HMA genes in Solanum tuberosum to elucidate their regulatory role in the mechanism governing the response of plants to abiotic stress.

Characterization of the Heavy-Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family from Triticeae Species.

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) only exist in vascular plants. They play important roles in responses to biotic/abiotic stresses, heavy-metal homeostasis, and detoxification. However, research on the distribution, diversification, and function of HIPPs in Triticeae species is limited. In this study, a total of 278 HIPPs were identified from a database from five Triticeae species, and 13 were cloned from Haynaldia villosa. These genes were classified into five groups by phylogenetic analysis. Most HIPPs had one HMA domain, while 51 from Clade I had two, and all HIPPs had good collinear relationships between species or subgenomes. In silico expression profiling revealed that 44 of the 114 wheat HIPPs were dominantly expressed in roots, 43 were upregulated under biotic stresses, and 29 were upregulated upon drought or heat treatment. Subcellular localization analysis of the cloned HIPPs from H. villosa showed that they were expressed on the plasma membrane. HIPP1-V was upregulated in H. villosa after Cd treatment, and transgenic wheat plants overexpressing HIPP1-V showed enhanced Cd tolerance, as shown by the recovery of seed-germination and root-growth inhibition by supplementary Cd. This research provides a genome-wide overview of the Triticeae HIPP genes and proved that HIPP1-V positively regulates Cd tolerance in common wheat.

Profiling of rice Cd-tolerant genes through yeast-based cDNA library survival screening

The bioaccumulation of cadmium (Cd) in crop and the subsequent food chain has aroused extensive concerns. However, the underlying molecular mechanisms of plant Cd tolerance remain to be clarified from the viewpoint of novel candidate genes. Here we described a highly efficient approach for preliminary identifying rice Cd-tolerant genes through the yeast-based cDNA library survival screening combined with high-throughput sequencing strategy. About 690 gene isoforms were identified as being Cd-tolerant candidates using this shotgun approach. Among the Cd-tolerant genes identified, several categories of genes such as BAX inhibitor (BI), NAC transcription factors and Rapid ALkalinization Factors (RALFs) were of particular interest, and their function of Cd tolerance was further validated via heterologous expression, which suggested that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves. Regarding the genes involved in ion transport, the validated Cd-tolerant heavy metal-associated domain (HMAD) isoprenylated protein HIPP42 was particularly noteworthy. Further elucidation of these genes associated with Cd tolerance in rice will benefit agricultural activities.

Heavy Metal Stress-Associated Proteins in Rice and Arabidopsis: Genome-Wide Identification, Phylogenetics, Duplication, and Expression Profiles Analysis.

Heavy metal exposure is a serious environmental stress in plants. However, plants have evolved several strategies to improve their heavy metal tolerance. Heavy metal-associated proteins (HMPs) participate in heavy metal detoxification. Here, we identified 46 and 55 HMPs in rice and Arabidopsis, respectively, and named them OsHMP 1–46 and AtHMP 1–55 according to their chromosomal locations. The HMPs from both plants were divided into six clades based on the characteristics of their heavy metal-associated domains (HMA). The HMP gene structures and motifs varied greatly among the different classifications. The HMPs had high collinearity and were segmentally duplicated. A cis-element analysis revealed that the HMPs may be regulated by different transcription factors. An expression profile analysis disclosed that only eight OsHMPs were constitutive in rice tissues. Of these, the expression of OsHMP37 was far higher than that of the other seven genes while OsHMP28 was expressed exclusively in the roots. For Arabidopsis, nine AtHMPs presented with very high transcript levels in all organs. Most of the selected OsHMPs were differentially expressed in various tissues under different heavy metal stresses. Only OsHMP09, OsHMP18, and OsHMP22 showed higher expression levels in all tissues under different heavy metal stresses. In contrast, most of the selected AtHMPs had nearly constant expression levels in different tissues under various heavy metal stresses. The AtHMP20, AtHMP23, AtHMP25, AtHMP31, AtHMP35, AtHMP46 expression levels under different heavy metal stresses were higher in the leaves and roots. The foregoing discoveries elucidated HMP evolution in monocotyledonous and dicotyledonous plants and may helpful functionally characterize HMPs in the future.

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

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.

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

Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice (Oryza sativa) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the effectors AVR–Pik and AVR–Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein–protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the “mis-matched” effector AVR–Pia in planta and that the Pikp–HMA domain binds AVR–Pia in vitro. We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR–Pia, and it does not trigger a plant response. The crystal structure of Pikp–HMA bound to AVR–Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR–Pik effectors, suggesting plasticity in integrated domain-effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles.

Specific recognition of two MAX effectors by integrated HMA domains in plant immune receptors involves distinct binding surfaces

The structurally conserved but sequence-unrelated MAX (Magnaporthe oryzae avirulence and ToxB-like) effectors AVR1-CO39 and AVR-PikD from the blast fungus M. oryzae are recognized by the rice nucleotide-binding domain and leucine-rich repeat proteins (NLRs) RGA5 and Pikp-1, respectively. This involves, in both cases, direct interaction of the effector with a heavy metal-associated (HMA) integrated domain (ID) in the NLR. Here, we solved the crystal structures of a C-terminal fragment of RGA5 carrying the HMA ID (RGA5_S), alone, and in complex with AVR1-CO39 and compared it to the structure of the Pikp1HMA/AVR-PikD complex. In both complexes, HMA ID/MAX effector interactions involve antiparallel alignment of β-sheets from each partner. However, effector-binding occurs at different surfaces in Pikp1HMA and RGA5HMA, indicating that these interactions evolved independently by convergence of these two MAX effectors to the same type of plant target proteins. Interestingly, the effector-binding surface in RGA5HMA overlaps with the surface that mediates RGA5HMA self-interaction. Mutations in the HMA-binding interface of AVR1-CO39 perturb RGA5HMA-binding, in vitro and in vivo, and affect the recognition of M. oryzae in a rice cultivar containing Pi-CO39 Our study provides detailed insight into the mechanisms of effector recognition by NLRs, which has substantial implications for future engineering of NLRs to expand their recognition specificities. In addition, we propose, as a hypothesis for the understanding of effector diversity, that in the structurally conserved MAX effectors the molecular mechanism of host target protein-binding is conserved rather than the host target proteins themselves.

Identification of a novel zinc-binding protein, C1orf123, as an interactor with a heavy metal-associated domain.

Heavy metal-associated (HMA) domains bind metal ions at its Cys-x-x-Cys (CxxC) motif and constitute an intracellular network for trafficking of metal ions for utilization and detoxification. We thus expect that novel metalloproteins can be identified by screening proteins interacting with a HMA domain. In this study, we performed yeast two-hybrid screening of the human proteome and found an uncharacterized protein encoded as open reading frame 123 in chromosome 1 (C1orf123) that can interact specifically with the HMA domain of a copper chaperone for superoxide dismutase (CCSdI). Our X-ray structural analysis of C1orf123 further revealed that it binds a Zn2+ ion in a tetrahedral coordination with four thiolate groups from two conserved CxxC motifs. For the interaction between C1orf123 and CCSdI, the CxxC motifs in both C1orf123 and CCSdI were required, implying metal-mediated interaction through the CxxC motifs. Notably, C1orf123 did not interact with several other HMA domains containing CxxC motifs, supporting high specificity in the interaction between C1orf123 and CCSdI. Based upon these results, we further discuss functional and structural significance of the interaction between C1orf123 and CCS.

Comparative analysis of Cd-responsive maize and rice transcriptomes highlights Cd co-modulated orthologs

BackgroundMetal tolerance is often an integrative result of metal uptake and distribution, which are fine-tuned by a network of signaling cascades and metal transporters. Thus, with the goal of advancing the molecular understanding of such metal homeostatic mechanisms, comparative RNAseq-based transcriptome analysis was conducted to dissect differentially expressed genes (DEGs) in maize roots exposed to cadmium (Cd) stress.ResultsTo unveil conserved Cd-responsive genes in cereal plants, the obtained 5166 maize DEGs were compared with 2567 Cd-regulated orthologs in rice roots, and this comparison generated 880 universal Cd-responsive orthologs groups composed of 1074 maize DEGs and 981 rice counterparts. More importantly, most of the orthologous DEGs showed coordinated expression pattern between Cd-treated maize and rice, and these include one large orthologs group of pleiotropic drug resistance (PDR)-type ABC transporters, two clusters of amino acid transporters, and 3 blocks of multidrug and toxic compound extrusion (MATE) efflux family transporters, and 3 clusters of heavy metal-associated domain (HMAD) isoprenylated plant proteins (HIPPs), as well as all 4 groups of zinc/iron regulated transporter protein (ZIPs). Additionally, several blocks of tandem maize paralogs, such as germin-like proteins (GLPs), phenylalanine ammonia-lyases (PALs) and several enzymes involved in JA biosynthesis, displayed consistent co-expression pattern under Cd stress.Out of the 1074 maize DEGs, approximately 30 maize Cd-responsive genes such as ZmHIPP27, stress-responsive NAC transcription factor (ZmSNAC1) and 9-cis-epoxycarotenoid dioxygenase (NCED, vp14) were also common stress-responsive genes reported to be uniformly regulated by multiple abiotic stresses. Moreover, the aforementioned three promising Cd-upregulated genes with rice counterparts were identified to be novel Cd-responsive genes in maize.Meanwhile, one maize glutamate decarboxylase (ZmGAD1) with Cd co-modulated rice ortholog was selected for further analysis of Cd tolerance via heterologous expression, and the results suggest that ZmGAD1 can confer Cd tolerance in yeast and tobacco leaves.ConclusionsThese novel findings revealed the conserved function of Cd-responsive orthologs and paralogs, which would be valuable for elucidating the genetic basis of the plant response to Cd stress and unraveling Cd tolerance genes.