RESUMEN

Plant defense and adaptation to adverse environmental conditions rely on gene expression control, such as mRNA transcription, processing, stability, and translation. Sudden temperature changes are common in the era of global warming; thus, understanding plant acclimation responses at the molecular level becomes imperative. mRNA translation initiation regulation has a pivotal role in achieving the synthesis of the appropriate battery of proteins needed to cope with temperature stress. In this study, we analyzed the role of translation initiation factors belonging to the eIF4E family in Arabidopsis acclimation to cold temperatures and freezing tolerance. Using knockout (KO) and overexpressing mutants of AteIF4E1 or AteIF(iso)4E, we found that AteIF4E1 but not AteIF(iso)4E overexpressing lines displayed enhanced tolerance to freezing without previous acclimation at 4°C. However, KO mutant lines, eif(iso)4e-1 and eif4e1-KO, were more sensitive to the stress. Cold acclimation in wild-type plants was accompanied by increased levels of eIF4E1 and eIF(iso)4E transcript levels, polysomes (P) enrichment, and shifts of these factors from translationally non-active to active fractions. Transcripts, previously found as candidates for eIF(iso)4E or eIF4E1 selective translation, changed their distribution in both P and total RNA in the presence of cold. Some of these transcripts changed their polysomal distribution in the mutant and one eIF4E1 overexpressing line. According to this, we propose a role of eIF4E1 and eIF(iso)4E in cold acclimation and freezing tolerance by regulating the expression of stress-related genes.

RESUMEN

The plant eukaryotic translation-initiation factors eIF4E and eIF(iso)4E play key roles in infection by plant RNA viruses, especially potyviruses. Mutations in the genes that encode these factors reduce susceptibility to the viruses. In the amphidiploid plant tobacco (Nicotiana tabacum L.), eIF4E1-S deletion mutants resist Potato virus Y (PVY), but resistance-breaking strains (RB-PVY) have appeared. In an earlier study, we demonstrated that the loss-of-function of eIF(iso)4E-T reduces susceptibility to RB-PVY. Here, we show that simultaneous inhibition of eIF4E1-S and eIF(iso)4E-T synergistically confers enhanced resistance to both PVY and RB-PVY without host growth or development defects. PVY symptoms and accumulation in a tobacco line lacking eIF4E1-S were detected at 14 days post-inoculation (dpi) and RB-PVY symptoms in lines without functional eIF(iso)4E-T were observed at 24 dpi. RB-PVY emerged in a PVY-infected tobacco line lacking eIF4E1-S. In contrast, lines without functional eIF4E1-S and eIF(iso)4E-T were nearly immune to PVY and RB-PVY, and little accumulation of either virus was detected even at 56 dpi. Thus, the lines will be promising for PVY-resistance breeding. This study provides a novel strategy to develop tobacco highly resistant to PVY and RB-PVY, and insights into the mechanisms responsible for high-level resistance.

RESUMEN

Plants protect against viruses through passive and active resistance mechanisms, and in most cases characterized thus far, natural recessive resistance to potyviruses has been mapped to mutations in the eukaryotic initiation factor eIF4E or eIF(iso)4E genes. Five eIF4E copies and three eIF(iso)4E copies were detected in Brassica rapa. The eIF4E and eIF(iso)4E genes could interact with turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) to initiate virus translation. From the yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays, the TuMV-CHN2/CHN3 VPgs could not interact with BraA.eIF4E.a/c or BraA.eIF(iso)4E.c, but they could interact with BraA.eIF(iso)4E.a in B. rapa. Further analysis indicated that the amino acid substitution L186F (nt T556C) in TuMV-UK1 VPg was important for the interaction networks between the TuMV VPg and eIF(iso)4E proteins. An interaction model of the BraA. eIF(iso)4E protein with TuMV VPg was constructed to infer the effect of the significant amino acids on the interaction of TuMV VPgs-eIF(iso)4Es, particularly whether the L186F in TuMV-UK1 VPg could change the structure of the TuMV-UK1 VPg protein, which may terminate the interaction of the BraA.eIF(iso)4E and TuMV VPg protein. This study provides new insights into the interactions between plant viruses and translation initiation factors to reveal the working of key amino acids.

RESUMEN

Tobacco bushy top disease (TBTD) is a viral disease of tobacco (Nicotiana tabacum L.) caused by mixed infection of Tobacco bushy top virus or Ethiopian tobacco bushy top virus and a helper virus. Despite its damage to tobacco, practical genetic resources for disease resistance have not been found. Here, we report that a mutation of tobacco eIF(iso)4E genes (eIF(iso)4E-S and eIF(iso)4E-T), which encode eukaryotic translation initiation factors, confers resistance (reduced susceptibility) to TBTD caused by a virus from Malawi (designated as tobacco bushy top virus Malawi isolate, TBTV-MW). RNAi lines in which eIF(iso)4E genes were silenced showed reduced susceptibility to TBTV-MW. We also tested chemically-induced single (eIF(iso)4E-S or eIF(iso)4E-T) and double (eIF(iso)4E-S and eIF(iso)4E-T) nonsense mutants for resistance to TBTV-MW. Suppression of eIF(iso)4E-S showed reduced susceptibility, and the resistance of the double mutant tended to be even stronger. eIF(iso)4E mutants also showed reduced susceptibility to TBTV-MW transmitted by aphids. To the best of our knowledge, the eIF(iso)4E-S mutant is the first genetic resource for TBTD resistance breeding in tobacco.

RESUMEN

RNA silencing and RNA decay are functionally interlaced, regulate gene expression and play a pivotal role in antiviral responses. As a counter-defensive strategy, many plant and mammalian viruses encode suppressors which interfere with both mechanisms. However, the protein interactions that connect these pathways remain elusive. Previous work reported that RNA silencing suppressors from different potyviruses, together with translation initiation factors EIF(iso)4E, interacted with the C-terminal region of the tobacco exoribonuclease RRP6-like 2, a component of the RNA decay exosome complex. Here, we investigate whether other viral silencing suppressors and cellular proteins might also bind RRP6-like exoribonucleases. A candidate search approach based on yeast two-hybrid protein interaction assays showed that three other unrelated viral suppressors, two from plant viruses and one from a mammalian virus, bound the C-terminus of the tobacco RRP6-like 2, the full-length of the Arabidopsis RRP6L1 protein and its C-terminal region. In addition, RRP6-like proteins were found to interact with members of the cellular double-stranded RNA-binding protein (DRB) family involved in RNA silencing. The C-terminal regions of RRP6L proteins are engaged in homotypic and heterotypic interactions and were predicted to be disordered. Collectively, these results suggest a protein interaction network that connects components of RNA decay and RNA silencing that is targeted by viral silencing suppressors.

Asunto(s)

Exorribonucleasas/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Virus de Plantas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Arabidopsis , Regulación de la Expresión Génica , Interacciones Microbiota-Huesped , Unión Proteica , Interferencia de ARN , Nicotiana

RESUMEN

Host proteins that are central to infection of potyviruses (genus Potyvirus; family Potyviridae) include the eukaryotic translation initiation factors eIF4E and eIF(iso)4E. The potyviral genome-linked protein (VPg) and the helper component proteinase (HCpro) interact with each other and with eIF4E and eIF(iso)4E and proteins are involved in the same functions during viral infection. VPg interacts with eIF4E/eIF(iso)4E via the 7-methylguanosine cap-binding region, whereas HCpro interacts with eIF4E/eIF(iso)4E via the 4E-binding motif YXXXXLΦ, similar to the motif in eIF4G. In this study, HCpro and VPg were found to interact in the nucleus, nucleolus, and cytoplasm in cells infected with the potyvirus potato virus A (PVA). In the cytoplasm, interactions between HCpro and VPg occurred in punctate bodies not associated with viral replication vesicles. In addition to HCpro, the 4E-binding motif was recognized in VPg of PVA. Mutations in the 4E-binding motif of VPg from PVA weakened interactions with eIF4E and heavily reduced PVA virulence. Furthermore, mutations in the 4G-binding domain of eIF4E reduced interactions with VPg and abolished interactions with HCpro. Thus, HCpro and VPg can both interact with eIF4E using the 4E-binding motif. Our results suggest a novel interaction network used by potyviruses to interact with host plants via translation initiation factors.

Asunto(s)

Interacciones Huésped-Patógeno , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Potyvirus/fisiología , Mapas de Interacción de Proteínas , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Núcleo Celular , Mutación , Fenotipo , Proteínas de Plantas/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Transporte de Proteínas , Nicotiana/virología , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Replicación Viral

RESUMEN

Eukaryotic translation-initiation factors eIF4E and eIF(iso)4E in plants play key roles in infection by potyviruses and other plant RNA viruses. Mutations in the genes encoding these factors reduce susceptibility to the viruses, and are the basis of several recessive virus resistance genes widely used in plant breeding. Because virus variants occasionally break such resistance, the molecular basis for this process must be elucidated. Although deletion mutants of eIF4E1-S of tobacco (Nicotiana tabacum L.) resist Potato virus Y (PVY; the type member of the genus Potyvirus), resistance-breaking strains of PVY threaten tobacco production worldwide. Here, we used RNA interference technology to knock down tobacco eIF4E2-S and eIF4E2-T genes or eIF(iso)4E-S and eIF(iso)4E-T genes. Transgenic plants with reduced transcript levels of both eIF(iso)4E-S and eIF(iso)4E-T showed reduced susceptibility to a resistance-breaking PVY strain with a K105E mutation in the viral genome-associated protein (VPg). By screening a population of chemically induced mutants of eIF(iso)4E-S and eIF(iso)4E-T, we showed that plants with a nonsense mutation in eIF(iso)4E-T, but not eIF(iso)4E-S, showed reduced susceptibility to the resistance-breaking PVY strain. In a yeast two-hybrid assay, VPg of the resistance-breaking strain, but not wild-type PVY, physically interacted with the eIF(iso)4E-T protein. Thus, eIF4E1-S is required for infection by PVY, but eIF(iso)4E-T is required for infection by the resistance-breaking strain. Our study provides the first evidence for the involvement of a host eukaryotic translation-initiation factor in the infection cycle of a resistance-breaking virus strain. The eIF(iso)4E-T mutants will be useful in tobacco breeding to introduce resistance against resistance-breaking PVY strains.

Asunto(s)

Nicotiana/virología , Enfermedades de las Plantas/virología , Potyvirus/patogenicidad , Mutación/genética

RESUMEN

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence-specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field-grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene-free T2 generation in self-pollinating species. Analysis of dry weights and flowering times for four independent T3 lines revealed no differences from wild-type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.

Asunto(s)

Arabidopsis/genética , Arabidopsis/virología , Sistemas CRISPR-Cas/genética , Resistencia a la Enfermedad/genética , Ingeniería Genética/métodos , Potyvirus/fisiología , Transgenes , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , Biomasa , Segregación Cromosómica/genética , Cruzamientos Genéticos , Electroforesis en Gel de Agar , Factor 4E Eucariótico de Iniciación/genética , Flores/fisiología , Edición Génica , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Sitio-Dirigida , Mutación/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , ARN Guía de Kinetoplastida/metabolismo

RESUMEN

The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.

Asunto(s)

Brassica rapa/genética , Brassica rapa/virología , Proteínas de Plantas/genética , Potyvirus/patogenicidad , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Resistencia a la Enfermedad/genética , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/genética , Genes de Plantas , Variación Genética , Interacciones Huésped-Patógeno/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Proteínas de Plantas/química , Plantas Modificadas Genéticamente , Potyvirus/genética , Conformación Proteica , Proteínas de Unión a Caperuzas de ARN/química , Proteínas de Unión a Caperuzas de ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética

RESUMEN

Recessive strain-specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad-spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis-splicing of the eIF(iso)4E allele in some TuMV-resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non-functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.

Asunto(s)

Brassica rapa/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Empalme del ARN , Brassica rapa/genética , Brassica rapa/virología , Codón de Terminación , Genes de Plantas , Genes Recesivos , Intrones , Datos de Secuencia Molecular