RESUMO
Establishment of arbuscular mycorrhiza relies on a plant signaling pathway that can be activated by fungal chitinic signals such as short-chain chitooligosaccharides and lipo-chitooligosaccharides (LCOs). The tomato LysM receptor-like kinase SlLYK10 has high affinity for LCOs and is involved in root colonization by arbuscular mycorrhizal fungi (AMF); however, its role in LCO responses has not yet been studied. Here, we show that SlLYK10 proteins produced by the Sllyk10-1 and Sllyk10-2 mutant alleles, which both cause decreases in AMF colonization and carry mutations in LysM1 and 2, respectively, have similar LCO-binding affinities compared to the WT SlLYK10. However, the mutant forms were no longer able to induce cell death in Nicotiana benthamiana when co-expressed with MtLYK3, a Medicago truncatula LCO co-receptor, while they physically interacted with MtLYK3 in co-purification experiments. This suggests that the LysM mutations affect the ability of SlLYK10 to trigger signaling through a potential co-receptor rather than its ability to bind LCOs. Interestingly, tomato lines that contain a calcium (Ca2+) concentration reporter [genetically encoded Ca2+ indicators (GECO)], showed Ca2+ spiking in response to LCO applications, but this occurred only in inner cell layers of the roots, while short-chain chitooligosaccharides also induced Ca2+ spiking in the epidermis. Moreover, LCO-induced Ca2+ spiking was decreased in Sllyk10-1*GECO plants, suggesting that the decrease in AMF colonization in Sllyk10-1 is due to abnormal LCO signaling.
Assuntos
Micorrizas , Proteínas de Plantas , Raízes de Plantas , Transdução de Sinais , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Micorrizas/fisiologia , Quitina/metabolismo , Lipopolissacarídeos/farmacologia , Oligossacarídeos/metabolismo , Mutação/genética , Regulação da Expressão Gênica de Plantas , Nicotiana/genética , Nicotiana/metabolismo , Quitosana/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/enzimologiaRESUMO
The establishment of the Legume-Rhizobia symbiosis is generally dependent on the production of rhizobial lipochitooligosaccharidic Nod factors (NFs) and their perception by plant Lysin Motif Receptor-Like Kinases (LysM-RLKs). In this study, we characterized a cluster of LysM-RLK genes implicated in strain-specific recognition in two highly divergent and widely-studied Medicago truncatula genotypes, A17 and R108. We then used reverse genetic approaches and biochemical analyses to study the function of selected genes in the clusters and the ability of their encoded proteins to bind NFs. Our study has revealed that the LYK cluster exhibits a high degree of variability among M. truncatula genotypes, which in A17 and R108 includes recent recombination events within the cluster and a transposon insertion in A17. The essential role of LYK3 in nodulation in A17 is not conserved in R108 despite similar sequences and good nodulation expression profiles. Although, LYK2, LYK5 and LYK5bis are not essential for nodulation of the two genotypes, some evidence points to accessory roles in nodulation, but not through high-affinity NF binding. This work shows that recent evolution in the LYK cluster provides a source of variation for nodulation, and potential robustness of signaling through genetic redundancy.
Assuntos
Medicago truncatula , Medicago truncatula/genética , Medicago truncatula/metabolismo , Família Multigênica , Simbiose/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Lysin motif receptor-like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defense. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high-affinity LCO-binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes, whole-genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO-binding characteristic and is dispensable for AM. Domain swapping between the three LysMs of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO-binding site is on the second LysM and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO-binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.
Assuntos
Medicago truncatula , Micorrizas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Micorrizas/metabolismo , Simbiose/genética , Quitina/metabolismoRESUMO
Symbiotic microorganisms such as arbuscular mycorrhizal fungi (AMF) produce both conserved microbial molecules that activate plant defense and lipo-chitooligosaccharides (LCOs) that modulate plant defense. Beside a well-established role of LCOs in the activation of a signaling pathway required for AMF penetration in roots, LCO perception and defense modulation during arbuscular mycorrhiza is not well understood. Here we show that members of the LYRIIIA phylogenetic group from the multigenic Lysin Motif Receptor-Like Kinase family have a conserved role in dicotyledons as modulators of plant defense and regulate AMF colonization in the Solanaceae species Nicotiana benthamiana. Interestingly, these proteins have a high-affinity for LCOs in plant species able to form a symbiosis with AMF but have lost this property in species that have lost this ability. Our data support the hypothesis that LYRIIIA proteins modulate plant defense upon LCO perception to facilitate AMF colonization in mycotrophic plant species and that only their role in plant defense, but not their ability to be regulated by LCOs, has been conserved in non-mycotrophic plants.
Assuntos
Quitosana , Micorrizas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Micorrizas/fisiologia , Quitosana/metabolismo , Quitina/metabolismo , Simbiose/fisiologia , Plantas/metabolismo , Raízes de Plantas/metabolismoRESUMO
Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.
Assuntos
Rhizobium leguminosarum , Rhizobium , Rhizobium leguminosarum/genética , Endófitos/genética , Triticum , Filogenia , Simbiose/genética , Bactérias/genética , Nódulos Radiculares de Plantas/microbiologiaRESUMO
N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.
Assuntos
Quitina , Medicago truncatula , Quitina/metabolismo , Quitosana , Medicago truncatula/genética , Simulação de Acoplamento Molecular , Oligossacarídeos , Tirosina/metabolismoRESUMO
Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis. In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM. RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels. Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.
Assuntos
Quitina/metabolismo , Lisina/metabolismo , Micorrizas/fisiologia , Imunidade Vegetal , Simbiose , Motivos de Aminoácidos , Sequência de Aminoácidos , Quitina/análogos & derivados , Quitinases/metabolismo , Quitosana , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inativação Gênica , Genes Fúngicos , Glomeromycota/genética , Glomeromycota/fisiologia , Interações Hospedeiro-Patógeno , Micélio/metabolismo , Micorrizas/genética , OligossacarídeosRESUMO
Bacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes.
Assuntos
Lipopolissacarídeos/metabolismo , Micorrizas/fisiologia , Rhizobium/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana , Fabaceae/metabolismo , Fabaceae/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Solanum lycopersicum/metabolismo , Micorrizas/metabolismo , Oligossacarídeos , Petunia/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Simbiose/genéticaRESUMO
Lipo-chitotetrasaccharide analogues where one central GlcNAc residue was replaced by a triazole unit have been synthesized from a derivative obtained by chitin depolymerization and a functionalized N-acetyl-glucosamine via the copper-catalyzed azide-alkyne cycloaddition. Their evaluation in a binding assay using LYR3, a putative lipo-chitooligosaccharide receptor in Medicago truncatula, shows a complete loss of binding.
Assuntos
Quitina/análogos & derivados , Medicago truncatula/química , Proteínas de Plantas/química , Quitina/síntese química , Quitina/química , Quitosana , OligossacarídeosRESUMO
LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.
Assuntos
Lipopolissacarídeos/metabolismo , Medicago truncatula/metabolismo , Proteínas Quinases/metabolismo , Protoplastos/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Medicago truncatula/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Simbiose , Nicotiana/genética , Nicotiana/metabolismoRESUMO
LYR3 [LysM (lysin motif) receptor-like kinase 3] of Medicago truncatula is a high-affinity binding protein for symbiotic LCO (lipo-chitooligosaccharide) signals, produced by rhizobia bacteria and arbuscular mycorrhizal fungi. The present study shows that LYR3 from several other legumes, but not from two Lupinus species which are incapable of forming the mycorrhizal symbiosis, bind LCOs with high affinity and discriminate them from COs (chitooligosaccharides). The biodiversity of these proteins and the lack of binding to the Lupinus proteins were used to identify features required for high-affinity LCO binding. Swapping experiments between each of the three LysMs of the extracellular domain of the M. truncatula and Lupinus angustifolius LYR3 proteins revealed the crucial role of the third LysM in LCO binding. Site-directed mutagenesis identified a tyrosine residue, highly conserved in all LYR3 LCO-binding proteins, which is essential for high-affinity binding. Molecular modelling suggests that it may be part of a hydrophobic tunnel able to accommodate the LCO acyl chain. The lack of conservation of these features in the binding site of plant LysM proteins binding COs provides a mechanistic explanation of how LCO recognition might differ from CO perception by structurally related LysM receptors.
Assuntos
Quitina/análogos & derivados , Medicago truncatula/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Quitina/metabolismo , Quitosana , Lupinus/metabolismo , Oligossacarídeos , Proteínas de Plantas/genética , Ligação Proteica , Transdução de Sinais , Simbiose/genética , Simbiose/fisiologiaRESUMO
PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses.
Assuntos
Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Micorrizas/fisiologia , Proteínas de Plantas/metabolismo , Rhizobium/fisiologia , Simbiose , Ubiquitinação , Contagem de Colônia Microbiana , Glomeromycota/fisiologia , Micorrizas/crescimento & desenvolvimento , Fosforilação , Proteínas de Plantas/química , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.
Assuntos
Quitina/análogos & derivados , Medicago truncatula/fisiologia , Oligossacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Lipídeos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Proteínas de Plantas/química , Alinhamento de Sequência , SimbioseRESUMO
The lysin motif receptor-like kinase, NFP (Nod factor perception), is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulfide bridges, and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulfide bridges involving CXC motifs in the extracellular region that are required for its biological function.
Assuntos
Membrana Celular/metabolismo , Medicago truncatula/citologia , Medicago truncatula/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , Retículo Endoplasmático/metabolismo , Glicosilação , Lisina , Medicago truncatula/fisiologia , Modelos Moleculares , Nodulação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de SinaisRESUMO
Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus/imunologia , Nucleotídeos/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Antivirais/imunologia , Arabidopsis/genética , Proteínas Argonautas , Inativação Gênica , Imunidade Inata/genética , Imunidade Inata/imunologia , Mutação/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Ligação ProteicaRESUMO
The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5'-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/embriologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Quimera/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Meristema/citologia , Meristema/metabolismo , MicroRNAs/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Estrutura Terciária de Proteína , Plântula/metabolismo , Sementes/citologia , Sementes/metabolismo , Supressão Genética , TransgenesRESUMO
The eukaryotic defense response posttranscriptional gene silencing (PTGS) is directed by short-interfering RNAs and thwarts invading nucleic acids via the RNA slicing activity of conserved ARGONAUTE (AGO) proteins. PTGS can be counteracted by exogenous or endogenous suppressors, including the cytoplasmic exoribonuclease XRN4, which also degrades microRNA (miRNA)-guided mRNA cleavage products but does not play an obvious role in development. Here, we show that the nuclear exoribonucleases XRN2 and XRN3 are endogenous PTGS suppressors. We also identify excised MIRNA loops as templates for XRN2 and XRN3 and show that XRN3 is critical for proper development. Independently, we identified the nucleotidase/phosphatase FIERY1 (FRY1) as an endogenous PTGS suppressor through a suppressor screen in a hypomorphic ago1 genetic background. FRY1 is one of six Arabidopsis thaliana orthologs of yeast Hal2. Yeast hal2 mutants overaccumulate 3'-phosphoadenosine 5'-phosphate, which suppresses the 5'-->3' exoribonucleases Xrn1 and Rat1. fry1 mutant plants recapitulate developmental and molecular characteristics of xrn mutants and likely restore PTGS in ago1 hypomorphic mutants by corepressing XRN2, XRN3, and XRN4, thus increasing RNA silencing triggers. We anticipate that screens incorporating partially compromised silencing components will uncover additional PTGS suppressors that may not be revealed using robust silencing systems.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Genes Supressores , Proteínas Nucleares/genética , Nucleotidases/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , Cucumovirus/fisiologia , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/química , MicroRNAs/metabolismo , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Nucleotidases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , TransgenesRESUMO
Plants contain more DICER-LIKE (DCL) enzymes and double-stranded RNA binding (DRB) proteins than other eukaryotes, resulting in increased small RNA network complexities. Analyses of single, double, triple and quadruple dcl mutants exposed DCL1 as a sophisticated enzyme capable of producing both microRNAs (miRNAs) and siRNAs, unlike the three other DCLs, which only produce siRNAs. Depletion of siRNA-specific DCLs results in unbalanced small RNA levels, indicating a redeployment of DCL/DRB complexes. In particular, DCL2 antagonizes the production of miRNAs and siRNAs by DCL1 in certain circumstances and affects development deleteriously in dcl1 dcl4 and dcl1 dcl3 dcl4 mutant plants, whereas dcl1 dcl2 dcl3 dcl4 quadruple mutant plants are viable. We also show that viral siRNAs are produced by DCL4, and that DCL2 can substitute for DCL4 when this latter activity is reduced or inhibited by viruses, pointing to the competitiveness of DCL2. Given the complexity of the small RNA repertoire in plants, the implication of each DCL, in particular DCL2, in the production of small RNAs that have no known function will constitute one of the next challenges.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Arabidopsis/virologia , Proteínas de Ciclo Celular/fisiologia , RNA Interferente Pequeno/biossíntese , Ribonuclease III/fisiologia , Ribonucleases/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , MicroRNAs/biossíntese , Vírus de Plantas/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonucleases/genéticaRESUMO
trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.