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
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
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
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.