RESUMO
The secondary metabolism of Piper species is known to produce a myriad of natural products from various biosynthetic pathways which, represent a rich source of previously uncharacterized chemical compounds. The determination of gene expression profiles in multiple tissue/organ samples could provide valuable clues towards understanding the potential biological functions of chemical changes in these plants. Studies on gene expression by RT-qPCR require particularly careful selection of suitable reference genes as a control for normalization. Here, we provide a study for the identification of reliable reference genes in P. arboreum, P. gaudichaudianum, P. malacophyllum, and P. tuberculatum, at two different life stages: 2-month-old seedlings and adult plants. To do this, annotated sequences were recovered from transcriptome datasets of the above listed Piper spp. These sequences were subjected to expression analysis using RT-qPCR, followed by analysis using the geNorm and NormFinder algorithms. A set of five genes were identified showing stable expression: ACT7 (Actin-7), Cyclophilin (Peptidyl-prolyl cis-trans isomerase), EF1α (Elongation factor 1-alpha), RNABP (RNA-binding protein), and UBCE (Ubiquitin conjugating enzyme). The universality of these genes was then validated using two target genes, ADC (arginine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase), which are involved in the biosynthesis of polyamines. We showed that normalization genes varied according to Piper spp., and we provide a list of recommended pairs of the best combination for each species. This study provides the first set of suitable candidate genes for gene expression studies in the four Piper spp. assayed, and the findings will facilitate subsequent transcriptomic and functional gene research.
Assuntos
Piper , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Piper/genética , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , TranscriptomaRESUMO
Innate immunity in plants relies on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the plant cell surface. CaLecRK-S.5, a pepper L-type lectin receptor kinase, has been shown to confer broad-spectrum resistance through priming activation. To further elucidate the molecular mechanism of CaLecRK-S.5, transgenic tobacco plants were generated in this study. Interestingly, hemizygous transgenic plants exhibited a high accumulation of CaLecRK-S.5, but this accumulation was completely abolished in homozygous transgenic plants by a cosuppression mechanism. Gain-of-function and loss-of-function analyses revealed that CaLecRK-S.5 plays a positive role in Phytophthora elicitin-mediated defense responses.
Assuntos
Nicotiana/imunologia , Phytophthora infestans/imunologia , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/imunologia , Proteínas Quinases/imunologia , Proteínas/imunologia , Piper/genética , Piper/imunologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Imunidade Vegetal , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Nicotiana/genética , TransgenesRESUMO
Piper colubrinum Link., a distant relative of Piper nigrum L., is immune to the oomycete pathogen Phytophthora capsici Leonian that causes 'quick wilt' in cultivated black pepper (P. nigrum). The osmotin, PR5 gene homologue, earlier identified from P. colubrinum, showed significant overexpression in response to pathogen and defense signalling molecules. The present study focuses on the functional validation of P. colubrinum osmotin (PcOSM) by virus induced gene silencing (VIGS) using Tobacco Rattle Virus (TRV)-based vector. P. colubrinum plants maintained under controlled growth conditions in a growth chamber were infiltrated with Agrobacterium carrying TRV empty vector (control) and TRV vector carrying PcOSM. Three weeks post infiltration, viral movement was confirmed in newly emerged leaves of infiltrated plants by RT-PCR using TRV RNA1 and TRV RNA2 primers. Semi-quantitative RT-PCR confirmed significant down-regulation of PcOSM gene in TRV-PcOSM infiltrated plant compared with the control plants. The control and silenced plants were challenged with Phytophthora capsici which demonstrated that knock-down of PcOSM in P. colubrinum leads to increased fungal mycelial growth in silenced plants compared to control plants, which was accompanied by decreased accumulation of H2O2 as indicated by 3,3'-diaminobenzidine (DAB) staining. Thus, in this study, we demonstrated that Piper colubrinum osmotin gene is required for resisting P. capsici infection and has possible role in hypersensitive cell death response and oxidative burst signaling during infection.
Assuntos
Inativação Gênica , Phytophthora/patogenicidade , Piper/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/genética , Vírus de Plantas/genética , Resistência à Doença/genética , Vetores Genéticos/genética , Piper/metabolismo , Piper/parasitologia , Piper/virologia , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismoRESUMO
In vitro activity of the essential oil from Piper diospyrifolium leaves was tested using disk diffusion techniques. The antifungal assay showed significant potencial antifungal activity: the oil was effective against several clinical fungal strains. The majority compounds in the essential oil were identified as sesquiterpenoids by GC-MS and GC-FID techniques.