RESUMO
Trichoderma atroviride is a root-colonizing fungus that confers multiple benefits to plants. In plants, small RNA (sRNA)-mediated gene silencing (sRNA-MGS) plays pivotal roles in growth, development, and pathogen attack. Here, we explored the role of core components of Arabidopsis thaliana sRNA-MGS pathways during its interaction with Trichoderma. Upon interaction with Trichoderma, sRNA-MGS-related genes paralleled the expression of Arabidopsis defense-related genes, linked to salicylic acid (SA) and jasmonic acid (JA) pathways. SA- and JA-related genes were primed by Trichoderma in leaves after the application of the well-known pathogen-associated molecular patterns flg22 and chitin, respectively. Defense-related genes were primed in roots as well, but to different extents and behaviors. Phenotypical characterization of mutants in AGO genes and components of the RNA-dependent DNA methylation (RdDM) pathway revealed that different sets of sRNA-MGS-related genes are essential for (i) the induction of systemic acquired resistance against Botrytis cinerea, (ii) the activation of the expression of plant defense-related genes, and (iii) root colonization by Trichoderma. Additionally, plant growth induced by Trichoderma depends on functional RdDM. Profiling of DNA methylation and histone N-tail modification patterns at the Arabidopsis Nitrile-Specifier Protein-4 (NSP4) locus, which is responsive to Trichoderma, showed altered epigenetic modifications in RdDM mutants. Furthermore, NSP4 is required for the induction of systemic acquired resistance against Botrytis and avoidance of enhanced root colonization by Trichoderma. Together, our results indicate that RdDM is essential in Arabidopsis to establish a beneficial relationship with Trichoderma. We propose that DNA methylation and histone modifications are required for plant priming by the beneficial fungus against B. cinerea.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Resistência à Doença/genética , Inativação Gênica , Hypocreales/genética , Nitrilas/metabolismo , RNA/metabolismo , Proteínas de Arabidopsis/metabolismo , Botrytis , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Hypocreales/metabolismo , Oxilipinas , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Raízes de Plantas/metabolismo , Ácido Salicílico/metabolismo , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Trichoderma spp. are filamentous fungi that colonize plant roots conferring beneficial effects to plants, either indirectly through the induction of their defense systems or directly through the suppression of phytopathogens in the rhizosphere. Transcriptomic analyses of Trichoderma spp. emerged as a powerful method for identifying the molecular events underlying the establishment of this beneficial relationship. Here, we focus on the transcriptomic response of Trichoderma virens during its interaction with Arabidopsis seedlings. The main response of T. virens to cocultivation with Arabidopsis was the repression of gene expression. The biological processes of transport and metabolism of carbohydrates were downregulated, including a set of cell wall-degrading enzymes putatively relevant for root colonization. Repression of such genes reached their basal levels at later times in the interaction, when genes belonging to the biological process of copper ion transport were induced, a necessary process providing copper as a cofactor for cell wall-degrading enzymes with the auxiliary activities class. RNA-Seq analyses showed the induction of a member of the SNF2 family of chromatin remodelers/helicase-related proteins, which was named IPA-1 (increased protection of Arabidopsis-1). Sequence analyses of IPA-1 showed its closest relatives to be members of the Rad5/Rad16 and SNF2 subfamilies; however, it grouped into a different clade. Although deletion of IPA-1 in T. virens did not affect its growth, the antibiotic activity of Δipa-1 culture filtrates against Rhizoctonia solani diminished but it remained unaltered against Botrytis cinerea. Triggering of the plant defense genes in plants treated with Δipa-1 was higher, showing enhanced resistance against Pseudomonas syringae but not against B. cinerea as compared with the wild type.
Assuntos
Antibiose , Arabidopsis/microbiologia , Montagem e Desmontagem da Cromatina , Resistência à Doença , Rhizoctonia/patogenicidade , Trichoderma/fisiologia , Humanos , Doenças das Plantas/microbiologia , Raízes de Plantas , TranscriptomaRESUMO
Plant microRNAs originate from a stem-loop structured single-stranded RNA precursor. Each stem-loop is processed to generate a mature microRNA that is recruited to an ARGONAUTE-containing multi-protein complex to direct silencing of its target mRNA. Here we report that the conserved plant miR159a precursor produces a second 21-nt long RNA with the properties of a microRNA. Its presence in different plant species is supported by its conservation in the stem-loop position and expression as determined by northern blot analysis. We show that successive processing by DCL1 produces this novel microRNA from the same precursor as miR159a. In contrast to the low levels observed in other plant models for the equivalent of miR159.2, in P. vulgaris, the accumulation of miR159.2 is easily detectable and when compared to miR159a, their expression patterns are distinct in different organs and growth conditions. Further evidence of the functionality of miR159.2 comes from its association with silencing complexes as demonstrated by co-immunoprecipitation experiments using an AGO1-specific antibody and processing of an artificial GFP reporter construct containing a complementary target sequence. These results indicate that the second small RNA corresponds to a microRNA, at least partially independent of miR159 activity, and that in plants a miRNA precursor may encode multiple regulatory small RNAs.
Assuntos
MicroRNAs/genética , Phaseolus/genética , Precursores de RNA/genética , RNA de Plantas/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Imunoprecipitação , MicroRNAs/metabolismo , Dados de Sequência Molecular , Oryza/genética , Phaseolus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Triticum/genéticaRESUMO
In plants, small RNA (microRNAs and other endogenous small RNAs)-guided target gene expression is vital for a wide variety of biological processes including adaptation to stress conditions. Identification of stress-regulated microRNAs or other classes of endogenous small RNAs advances our understanding of post-transcriptional gene regulation important for plant stress tolerance. This chapter describes a detailed step-by-step protocol for cloning of small RNAs. Following 5' and 3' adapter ligation to the purified small RNAs, cDNA will be synthesized using reverse transcription, which will be further amplified using a polymerase chain reaction. The resulting small DNA fragments can be either cloned and sequenced using traditional sequencing method or subjected to direct high-throughput pyrosequencing or sequencing-by-synthesis technology.
Assuntos
Clonagem Molecular/métodos , MicroRNAs/genética , RNA de Plantas/genética , Estresse Fisiológico/genética , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica de Plantas , MicroRNAs/isolamento & purificação , Desnaturação de Ácido Nucleico/genética , Phaseolus/genética , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues.