RESUMO
RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.
Assuntos
Nicotiana/genética , Nicotiana/microbiologia , Proteínas de Plantas/genética , Pseudomonas fluorescens , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Algoritmos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Folhas de Planta/genética , Folhas de Planta/microbiologia , Pseudomonas fluorescens/patogenicidadeRESUMO
The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.
Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Pseudomonas syringae , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Transcriptoma , Algoritmos , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Reprodutibilidade dos Testes , Análise de Sequência de RNARESUMO
The complete genomic sequence of a new tobamovirus in tomatoes was determined through deep sequencing and assembly of small RNAs, then validated through Sanger sequencing. Based on the low sequence identity (≤85%) to known viruses and a close phylogenetic relationship to tobamoviruses, it was identified as a new species.
RESUMO
Impaired root development caused by aluminum (Al) toxicity is a major cause of grain yield reduction in crops cultivated on acid soils, which are widespread worldwide. In sorghum, the major Al-tolerance locus, AltSB , is due to the function of SbMATE, which is an Al-activated root citrate transporter. Here we performed a molecular and physiological characterization of various AltSB donors and near-isogenic lines harboring various AltSB alleles. We observed a partial transfer of Al tolerance from the parents to the near-isogenic lines that was consistent across donor alleles, emphasizing the occurrence of strong genetic background effects related to AltSB . This reduction in tolerance was variable, with a 20% reduction being observed when highly Al-tolerant lines were the AltSB donors, and a reduction as great as 70% when other AltSB alleles were introgressed. This reduction in Al tolerance was closely correlated with a reduction in SbMATE expression in near-isogenic lines, suggesting incomplete transfer of loci acting in trans on SbMATE. Nevertheless, AltSB alleles from the highly Al-tolerant sources SC283 and SC566 were found to retain high SbMATE expression, presumably via elements present within or near the AltSB locus, resulting in significant transfer of the Al-tolerance phenotype to the derived near-isogenic lines. Allelic effects could not be explained by coding region polymorphisms, although occasional mutations may affect Al tolerance. Finally, we report on the extensive occurrence of alternative splicing for SbMATE, which may be an important component regulating SbMATE expression in sorghum by means of the nonsense-mediated RNA decay pathway.
Assuntos
Alumínio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Sorghum/efeitos dos fármacos , Sorghum/metabolismo , Processamento Alternativo , Sequência de Bases , Variação Genética , Genoma de Planta , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/genética , Conformação Proteica , Sorghum/genéticaRESUMO
Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5-7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.