RESUMO
To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.
Assuntos
Basidiomycota , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Basidiomycota/genética , Fungos/genética , Pirofosfatases/metabolismo , Virulência/genética , Doenças das Plantas/microbiologia , Nudix HidrolasesRESUMO
We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane-anchored leucine-rich repeat receptor-like protein (LRR-RLP). Unlike most other LRR-RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR-RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR-RLPs, recognition specificity is determined in the C-terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1-dependent necrosis in Nicotiana benthamiana depends on the LRR receptor-like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR-RLPs involved in plant defence all carry residues in their last LRR and C-terminal LRR capping domain that are conserved with SERK3/BAK1-interacting residues in the same relative positions in the LRR-RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1-dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.
Assuntos
Fusarium/patogenicidade , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Proteínas de Repetições Ricas em Leucina , Solanum lycopersicum/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas/genéticaRESUMO
BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.
Assuntos
Basidiomycota/genética , Mapeamento Cromossômico , Genoma Fúngico , Genômica , Virulência/genética , Sequência de Aminoácidos , Basidiomycota/patogenicidade , Biologia Computacional/métodos , Frequência do Gene , Loci Gênicos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Perda de Heterozigosidade , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Recombinação GenéticaRESUMO
Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.
Assuntos
Basidiomycota/imunologia , Linho/imunologia , Proteínas Fúngicas/imunologia , Doenças das Plantas/imunologia , Sequência de Aminoácidos , Basidiomycota/genética , Basidiomycota/fisiologia , Cristalografia por Raios X , Linho/citologia , Linho/microbiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Immunoblotting , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosfatidilinositóis/imunologia , Fosfatidilinositóis/metabolismo , Células Vegetais/imunologia , Células Vegetais/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica/imunologia , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plant resistance proteins provide race-specific immunity through the recognition of pathogen effectors. The resistance genes I, I-2 and I-3 have been incorporated into cultivated tomato (Solanum lycopersicum) from wild tomato species to confer resistance against Fusarium oxysporum f. sp. lycopersici (Fol) races 1, 2 and 3, respectively. Although the Fol effectors corresponding to these resistance genes have all been identified, only the I-2 resistance gene has been isolated from tomato. To isolate the I-3 resistance gene, we employed a map-based cloning approach and used transgenic complementation to test candidate genes for resistance to Fol race 3. Here, we describe the fine mapping and sequencing of genes at the I-3 locus, which revealed a family of S-receptor-like kinase (SRLK) genes. Transgenic tomato lines were generated with three of these SRLK genes and one was found to confer Avr3-dependent resistance to Fol race 3, confirming it to be I-3. The finding that I-3 encodes an SRLK reveals a new pathway for Fol resistance and a new class of resistance genes, of which Pi-d2 from rice is also a member. The identification of I-3 also allows the investigation of the complex effector-resistance protein interaction involving Avr1-mediated suppression of I-2- and I-3-dependent resistance in tomato.
Assuntos
Resistência à Doença/genética , Fusarium/fisiologia , Genes de Plantas , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Agrobacterium/fisiologia , Sequência de Aminoácidos , Pareamento de Bases , Morte Celular , Membrana Celular/metabolismo , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Estudos de Associação Genética , Loci Gênicos , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Família Multigênica , Mapeamento Físico do Cromossomo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Alinhamento de Sequência , Nicotiana/citologiaRESUMO
In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M-AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes.
Assuntos
Basidiomycota/fisiologia , Linho/microbiologia , Doenças das Plantas/microbiologia , Sequência de Aminoácidos , Aminoácidos/genética , Basidiomycota/genética , Basidiomycota/metabolismo , Morte Celular , Linho/genética , Linho/metabolismo , Imunidade Inata , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.
Assuntos
Basidiomycota/genética , Basidiomycota/patogenicidade , Linho/genética , Linho/microbiologia , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/microbiologia , Fatores de Virulência/genética , Biologia Computacional , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Micoses/genética , Micoses/microbiologia , Doenças das Plantas/genética , Folhas de Planta/microbiologia , Esporos Fúngicos/genética , Transcriptoma/fisiologia , Virulência/genéticaRESUMO
Recent advances in cloning avirulence genes from a rust fungus and three oomycete species have provided the novel insight that these eukaryotic plant pathogens deliver small proteins into the host cell cytoplasm where they are recognized by resistance proteins. Anne Rehmany et al. have recently identified a potential host-targeting signal in oomycete avirulence proteins from Hyaloperonospora parasitica, Phytophthora sojae and Phytophthora infestans that might be involved in transporting proteins into the host cell. This signal is surprisingly similar to the host targeting signal used by the malaria pathogen Plasmodium fulciparum to target virulence proteins to the mammalian host cell.
Assuntos
Proteínas de Algas/fisiologia , Oomicetos/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Algas/metabolismo , Animais , Arabidopsis , Basidiomycota/genética , Basidiomycota/fisiologia , Citoplasma/microbiologia , Linho , Oomicetos/genética , Phytophthora/genética , Phytophthora/fisiologia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Transporte Proteico , Transdução de Sinais , Solanum tuberosum , Glycine maxRESUMO
RNA sequencing (RNAseq) reads from cape gooseberry plants (Physalis peruviana) infected with Fusarium oxysporumf. sp. physali (Foph) were mapped against the lineage-specific transcriptome of Fusarium oxysporumf. sp. lycopersici (Fol) to look for putative effector genes. Homologues of Fol SIX1(designated SIX1a and SIX1b), SIX7, SIX10, SIX12, SIX15 and Ave1were identified. The near identity of the Foph and Fol SIX7, SIX10 and SIX12genes and their intergenic regions suggest that this gene cluster may have undergone recent lateral transfer. Foph SIX1a and SIX1bwere tested for their ability to complement a SIX1 knockout mutant of Fol. This mutant shows reduced pathogenicity on susceptible tomato plants, but is able to infect otherwise resistant tomato plants carrying the I-3 gene for Fusarium wilt resistance (SIX1 corresponds to Avr3). Neither SIX1a nor SIX1b could restore full pathogenicity on susceptible tomato plants, suggesting that any role they may play in pathogenicity is likely to be specific to cape gooseberry. SIX1b, but not SIX1a, was able to restore avirulence on tomato plants carrying I-3.These findings separate the recognition of SIX1 from its role as an effector and suggest direct recognition by I-3. A hypervariable region of SIX1undergoing diversifying selection within the F. oxysporum species complex is likely to play an important role in SIX1 recognition. These findings also indicate that I-3could potentially be deployed as a transgene in cape gooseberry to protect this emerging crop from Foph.Alternatively, cape gooseberry germplasm could be explored for I-3homologues capable of providing resistance to Foph.
Assuntos
Fusarium/patogenicidade , Transferência Genética Horizontal/genética , Physalis/microbiologia , Doenças das Plantas/microbiologia , Solanum lycopersicum/microbiologia , Proteínas Fúngicas/genética , Fusarium/genéticaRESUMO
The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.
Assuntos
Basidiomycota/metabolismo , Núcleo Celular/metabolismo , Resistência à Doença , Linho/microbiologia , Proteínas Fúngicas/química , Proteínas de Plantas/metabolismo , Agrobacterium/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Células Vegetais/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Nicotiana/genética , Zinco/metabolismoRESUMO
A major insight that has emerged in the study of haustoria-forming plant pathogens over the last few years is that these eukaryotic biotrophs deliver suites of secreted proteins into host cells during infection. This insight has largely derived from successful efforts to identify avirulence (Avr) genes and their products from these pathogens. These Avr genes, identified from a rust and a powdery mildew fungus and three oomycete species, encode small proteins that are recognized by resistance proteins in the host plant cytoplasm, suggesting that they are transported inside plant cells during infection. These Avr proteins probably represent examples of fungal and oomycete effector proteins with important roles in subverting host cell biology during infection. In this respect, they represent a new opportunity to understand the basis of disease caused by these biotrophic pathogens. Elucidating how these pathogen proteins gain entry into plant cells and their biological function will be key questions for future research.
Assuntos
Proteínas de Algas/metabolismo , Ascomicetos/patogenicidade , Basidiomycota/patogenicidade , Membrana Celular/ultraestrutura , Proteínas Fúngicas/metabolismo , Oomicetos/patogenicidade , Proteínas de Algas/genética , Ascomicetos/genética , Basidiomycota/genética , Membrana Celular/microbiologia , Proteínas Fúngicas/genética , Oomicetos/genética , Doenças das Plantas/microbiologia , Plantas/microbiologia , Virulência/genéticaRESUMO
Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 A) is compatible with the Cu K alpha wavelength (1.54 A) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.
Assuntos
Basidiomycota/química , Cobalto/química , Cristalografia por Raios X/métodos , Linho/microbiologia , Proteínas Fúngicas/química , Basidiomycota/patogenicidade , Cristalização , Linho/química , Doenças das Plantas/microbiologia , Virulência/fisiologiaRESUMO
Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/.
Assuntos
Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Software , Sequência de Aminoácidos , Genoma Fúngico , Oomicetos/metabolismo , Organelas/metabolismo , Proteínas de Plantas/química , Sinais Direcionadores de Proteínas , Transporte Proteico , Frações Subcelulares/metabolismo , Nicotiana/metabolismoRESUMO
In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Fusarium/patogenicidade , Hormônios Peptídicos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Fúngicas/genética , Fusarium/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Hormônios Peptídicos/genética , Proteínas de Plantas/genética , Plântula/genética , Plântula/metabolismo , Plântula/microbiologia , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
The tomato I-3 and I-7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I-3 has been identified previously on chromosome 7 and encodes an S-receptor-like kinase, but little is known about I-7. Molecular markers have been developed for the marker-assisted breeding of I-3, but none are available for I-7. We used an RNA-seq and single nucleotide polymorphism (SNP) analysis approach to map I-7 to a small introgression of S. pennelliiâ DNA (c. 210 kb) on chromosome 8, and identified I-7 as a gene encoding a leucine-rich repeat receptor-like protein (LRR-RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I-7, like many other LRR-RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I-7 gene for Fol resistance, we found that I-7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I-7-mediated resistance, unlike I-2- or I-3-mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1-independent pathway for resistance activation. The identification of I-7 has allowed us to develop molecular markers for marker-assisted breeding of both genes currently known to confer Fol race 3 resistance (I-3 and I-7). Given that I-7-mediated resistance is not suppressed by Avr1, I-7 may be a useful addition to I-3 in the tomato breeder's toolbox.
Assuntos
Resistência à Doença/genética , Fusarium/fisiologia , Genes de Plantas , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Alelos , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Sequência Conservada/genética , Família Multigênica , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de RNARESUMO
Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.
Assuntos
Endopeptidases/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Vetores Genéticos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Cloreto de Sódio , Ubiquitina/genética , Proteases Específicas de UbiquitinaRESUMO
Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.
Assuntos
Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ubiquitina/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Carioferinas/química , Carioferinas/isolamento & purificação , Carioferinas/metabolismo , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/isolamento & purificação , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Ubiquitina/químicaRESUMO
The haustorium is a distinguishing feature of biotrophic plant pathogens. Several highly diverged -pathogen classes have independently evolved haustoria, suggesting that they represent an effective adaptation for growing within living plant tissue. Despite their clear importance in biotrophy, they have been difficult to study due to the close association of biotrophic pathogens with their host and the inability to produce haustoria in vitro. These drawbacks have been circumvented in the study of rust fungi by the development of a haustoria isolation technique. The strong binding of the lectin concanavalin A (ConA) to rust haustoria allows these structures to be purified from infected plant tissue by affinity chromatography on a ConA-Sepharose macrobead column. The isolation process results in substantial yields of intact haustoria that retain their cytoplasmic contents, making them amenable to experimentation. The construction of cDNA libraries from isolated rust haustoria and their subsequent sequence analysis have provided significant insight into haustoria function at a molecular level, revealing important roles in nutrient acquisition and the delivery of pathogenicity effector proteins. The generation of a rust haustorium-specific cDNA library is described in this chapter.
Assuntos
Basidiomycota/genética , Basidiomycota/isolamento & purificação , Biblioteca Gênica , Plantas/microbiologia , Basidiomycota/patogenicidade , Basidiomycota/ultraestrutura , Cromatografia/instrumentação , Cromatografia/métodosRESUMO
The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.
Assuntos
Basidiomycota/química , Basidiomycota/patogenicidade , Linho/microbiologia , Imunidade Inata/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Fatores de Virulência/química , Sequência de Aminoácidos , Cristalografia por Raios X , Análise Mutacional de DNA , Linho/química , Linho/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Fatores de Virulência/metabolismoRESUMO
Rust fungi, obligate biotrophs that cause disease and yield losses in crops such as cereals and soybean (Glycine max), obtain nutrients from the host through haustoria, which are specialized structures that develop within host cells. Resistance of flax (Linum usitatissimum) to flax rust (Melampsora lini) involves the induction of a hypersensitive cell death response at haustoria formation sites, governed by gene-for-gene recognition between host resistance and pathogen avirulence genes. We identified genes encoding haustorially expressed secreted proteins (HESPs) by screening a flax rust haustorium-specific cDNA library. Among 429 unigenes, 21 HESPs were identified, one corresponding to the AvrL567 gene. Three other HESPs cosegregated with the independent AvrM, AvrP4, and AvrP123 loci. Expression of these genes in flax induced resistance gene-mediated cell death with the appropriate specificity, confirming their avirulence activity. AvrP4 and AvrP123 are Cys-rich proteins, and AvrP123 contains a Kazal Ser protease inhibitor signature, whereas AvrM contains no Cys residues. AvrP4 and AvrM induce cell death when expressed intracellularly, suggesting their translocation into plant cells during infection. However, secreted AvrM and AvrP4 also induce necrotic responses, with secreted AvrP4 more active than intracellular AvrP4, possibly as a result of enhanced formation of endoplasmic reticulum-dependent disulfide bonds. Addition of an endoplasmic reticulum retention signal inhibited AvrM-induced necrosis, suggesting that both AvrM and AvrP4 can reenter the plant cell after secretion in the absence of the pathogen.