RESUMO
In animals and plants, innate immunity is regulated by nucleotide binding domain and leucine-rich repeat (NB-LRR) proteins that mediate pathogen recognition and that activate host-cell defense responses. Plant NB-LRR proteins, referred to as R proteins, have amino-terminal domains that contain a coiled coil (CC) or that share similarity with animal Toll and interleukin 1 receptors (TIR). To investigate R protein function, we are using the TIR-NB-LRR protein N that mediates resistance against tobacco mosaic virus (TMV) through recognition of the TMV p50 protein. Here, we describe N requirement gene 1 (NRG1), a novel N-resistance component that was identified by a virus-induced gene silencing (VIGS) screen of a cDNA library. Surprisingly, NRG1 encodes an NB-LRR type R protein that, in contrast to N, contains a CC rather than a TIR domain. Our findings support emerging evidence that many disease-resistance pathways each recruit more than a single NB-LRR protein. The results also indicate that, in addition to the previously recognized role in elicitor recognition, NB-LRR proteins may also function in downstream signaling pathways.
Assuntos
Imunidade Inata/imunologia , Nicotiana/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Transdução de Sinais/imunologia , Vírus do Mosaico do Tabaco , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Componentes do Gene , Biblioteca Gênica , Inativação Gênica , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Nicotiana/virologiaRESUMO
SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Imunidade , Proteínas de Plantas/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Arabidopsis/imunologia , Sequência Conservada , Genes Dominantes , Imunidade Inata , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/química , Potexvirus/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Solanum tuberosum/imunologia , Soluções , Nicotiana/virologiaRESUMO
A highly conserved eukaryotic protein SGT1 binds specifically to the molecular chaperone, HSP90. In plants, SGT1 positively regulates disease resistance conferred by many Resistance (R) proteins and developmental responses to the phytohormone, auxin. We show that silencing of SGT1 in Nicotiana benthamiana causes a reduction in steady-state levels of the R protein, Rx. These data support a role of SGT1 in R protein accumulation, possibly at the level of complex assembly. In Arabidopsis, two SGT1 proteins, AtSGT1a and AtSGT1b, are functionally redundant early in development. AtSGT1a and AtSGT1b are induced in leaves upon infection and either protein can function in resistance once a certain level is attained, depending on the R protein tested. In unchallenged tissues, steady-state AtSGT1b levels are at least four times greater than AtSGT1a. While the respective tetratricopeptide repeat (TPR) domains of SGT1a and SGT1b control protein accumulation, they are dispensable for intrinsic functions of SGT1 in resistance and auxin responses.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Proteínas de Ciclo Celular/fisiologia , Nicotiana/imunologia , Doenças das Plantas/genética , Proteínas de Plantas/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/química , Folhas de Planta/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Estrutura Terciária de Proteína/genética , Pseudomonas syringae , Sementes/genética , Sementes/metabolismo , Deleção de Sequência , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx lacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions.
Assuntos
Proteínas do Capsídeo , Proteínas de Plantas/química , Solanum tuberosum/metabolismo , Motivos de Aminoácidos , Capsídeo/química , Capsídeo/metabolismo , Inativação Gênica , Teste de Complementação Genética , Imunidade Inata/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potexvirus/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Solanum tuberosum/genética , Solanum tuberosum/virologiaRESUMO
Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.
Assuntos
Inativação Gênica , Engenharia Genética/métodos , Vírus de Plantas/genética , Plantas/genética , RNA Viral , Vetores GenéticosRESUMO
In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide-binding site, leucine-rich-repeats and amino-terminal similarity to animal Toll and Interleukin-1 (so-called TIR-NBS-LRR proteins). EDS1 is not required by NBS-LRR proteins that possess an amino-terminal coiled-coil motif (CC-NBS-LRR proteins). Using virus-induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR-NBS-LRR protein, was EDS1-dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC-NBS-LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR-NBS-LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.
Assuntos
Proteínas de Arabidopsis , Proteínas de Ligação a DNA/genética , Nicotiana/genética , Proteínas de Plantas/genética , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/virologia , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Inativação Gênica , Imunidade Inata/genética , Zíper de Leucina/genética , Lipase/genética , Lipase/metabolismo , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Vírus de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Rhizobium/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Nicotiana/virologiaRESUMO
The Lycopersicon esculentum Bs4 resistance (R) gene specifies recognition of Xanthomonas campestris pv. vesicatoria (Xcv) strains that express the cognate AvrBs4 avirulence protein. Bs4 was isolated by positional cloning and is predicted to encode a nucleotide-binding leucine-rich repeat (NB-LRR) protein that is homologous to tobacco N and potato Y-1 resistance proteins. Xcv infection tests demonstrate that Bs4 confers perception of AvrBs4 but not the 97% identical AvrBs3 protein. However, when delivered via Agrobacterium T-DNA transfer, both, avrBs4 and avrBs3 trigger a Bs4-dependent hypersensitive response, indicating that naturally occurring AvrBs3-homologues provide a unique experimental platform for molecular dissection of recognition specificity. Transcript studies revealed intron retention in Bs4 transcripts. Yet, an intron-deprived Bs4 derivative still mediates AvrBs4 detection, suggesting that the identified splice variants are not crucial to resistance. The L. pennellii bs4 allele, which is >98% identical to L. esculentum Bs4, has a Bs4-like exon-intron structure with exception of a splice polymorphism in intron 2 that causes truncation of the predicted bs4 protein. To test if the receptor-ligand model is a valid molecular description of Bs4-mediated AvrBs4 perception, we conducted yeast two-hybrid studies. However, a direct interaction was not observed. Defense signaling of the Bs4-governed reaction was studied in Nicotiana benthamiana by virus-induced gene silencing and showed that Bs4-mediated resistance is EDS1- and SGT1-dependent.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Agrobacterium tumefaciens/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Imunidade Inata/genética , Proteínas de Repetições Ricas em Leucina , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Dados de Sequência Molecular , Mutação , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
It has been suggested that, in addition to viral proteins, host proteins are involved in RNA virus replication. In this study the RNA helicase domain of the Tobacco mosaic virus (TMV) replicase proteins was used as bait in the yeast two-hybrid system to identify tobacco proteins with a putative role in TMV replication. Two host proteins were characterized. One protein (designated #3) belongs to a protein family of ATPases associated with various activities (AAA), while the second host protein (designated #13) is the 33K subunit of the oxygen-evolving complex of photosystem II. Using Tobacco rattle virus vectors, genes #3 and #13 were silenced in Nicotiana benthamiana, after which the plants were challenged by TMV infection. Silencing of gene #13 resulted in a 10-fold increase of TMV accumulation, whereas silencing of gene #3 caused a twofold reduction of TMV accumulation. Additionally, silencing of genes #3 and #13 decreased and increased, respectively, the accumulation of two other viruses. Similar to silencing of gene #13, inhibition of photosystem II by application of an herbicide increased TMV accumulation several fold. Infection of N. benthamiana with TMV resulted in a decrease of #13 mRNA levels. Silencing of gene #13 may reflect a novel strategy of TMV to suppress basal host defense mechanisms. The two-hybrid screenings did not identify tobacco proteins involved in helicase domain-induced N-mediated resistance.
Assuntos
Inativação Gênica , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Proteínas de Plantas/genética , RNA Helicases/metabolismo , Vírus do Mosaico do Tabaco/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Diurona/farmacologia , Herbicidas/farmacologia , Dados de Sequência Molecular , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Doenças das Plantas/virologia , Folhas de Planta/virologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/patogenicidade , Técnicas do Sistema de Duplo-Híbrido , Replicação ViralRESUMO
Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants.
Assuntos
Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , DNA de Plantas/genética , Genes de Plantas , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Potexvirus/patogenicidade , Proteínas Serina-Treonina Quinases/fisiologia , Pseudomonas syringae/patogenicidade , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/microbiologia , Nicotiana/virologia , Vírus do Mosaico do Tabaco/patogenicidadeRESUMO
The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.
Assuntos
Proteínas de Transporte/metabolismo , Hordeum/genética , Hordeum/metabolismo , Imunidade Inata/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo/genética , Fungos/fisiologia , Homeostase/fisiologia , Hordeum/microbiologia , Interações Hospedeiro-Parasita/fisiologia , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Regulação para Cima/genéticaRESUMO
Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.