RESUMO
WRKY transcription factors are key regulators that activate and fine-tune stress responses, including defense responses against pathogens. We isolated a poplar (Populus tremulaxPopulus alba) cDNA sequence, PtWRKY23, that encodes the ortholog of Arabidopsis WRKY23 and present the functional analysis of WRKY23, with emphasis on its potential role in resistance to rust infection. To investigate the function of PtWRKY23, we examined PtWRKY23 expression after stress treatments by qRT-PCR and generated PtWRKY23-misexpressing plants. Transgenic plants were assessed for resistance to Melampsora rust and were analyzed using the poplar Affymetrix GeneChip and histological techniques to study the consequences of PtWRKY23 misexpression. PtWRKY23 is rapidly induced by Melampsora infection and elicitor treatments and poplars overexpressing and underexpressing PtWRKY23 were both more susceptible to Melampsora infection than wild type. Transcriptome analysis of PtWRKY23 overexpressors revealed a significant overlap with the Melampsora-infection response. Transcriptome analysis also suggests that PtWRKY23 affects redox homeostasis and cell wall-related metabolism, which was confirmed by analyses that showed that PtWRKY23-misexpressing plants have altered peroxidase activity, apparent H(2)O(2) accumulation and lignin deposition. Our results show that PtWRKY23 affects resistance to Melampsora infection and that this may be caused by deregulation of genes that disrupt redox homeostasis and cell wall metabolism.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/genética , Populus/imunologia , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Quitosana/farmacologia , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Peroxidase/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Populus/efeitos dos fármacos , Populus/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Salicílico/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Madeira/efeitos dos fármacos , Madeira/genéticaRESUMO
In natural conditions, plants are subjected to a combination of biotic stresses and often have to cope with simultaneous pathogen infections. In this report, we aim to understand the global transcriptional response of hybrid poplar NM6 (Populus nigra x P. maximowiczii) to infection by two biotrophic Melampsora fungi, Melampsora larici-populina and M. medusae f. sp. deltoidae. These pathogens triggered different responses after inoculation of poplar leaves. Transcript profiling using the GeneChip Poplar Genome Array revealed a total of 416 differentially expressed transcripts whose expression level was > or = twofold relative to controls. Interestingly, approximately half of the differentially expressed genes in infected leaves showed altered expression following interaction with either of the Melampsora spp. We also infected poplar leaves simultaneously with both Melampsora spp. to investigate potential interaction between the responses to the individual pathogens during a mixed infection. For this mixed inoculation, the number of differentially expressed transcripts increased to 648 and our analysis showed that infection with both fungi also induced a common set of genes. The genes induced after Melampsora spp. infection were mainly related to primary and secondary metabolic processes, cell-wall reinforcement and lignification, defense and stress-related mechanisms, and signal perception and transduction.
Assuntos
Basidiomycota/fisiologia , Quimera/metabolismo , Perfilação da Expressão Gênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Populus/genética , Populus/microbiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Anti-Infecciosos/metabolismo , Parede Celular/genética , Células Clonais , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/genética , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase , Populus/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
The gene coding for green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully used as a vital marker for the transformation of three woody plant species, black spruce (Picea mariana (Mill.) BSP), white pine (Pinus strobus L.) and poplar (Populus spp.). The gfp gene and the gene conferring resistance to kanamycin (nptII) were introduced by microprojectile bombardment or Agrobacterium tumefaciens-mediated technology. Screening by fluorescence microscopy of the transformed plant material, under the selection of kanamycin, identified five to eight cell lines from each tree species that clearly expressed GFP. Expression of GFP was observed in somatic embryonal cells of the coniferous species and in stem sections of poplar. For all species, GFP transgene expression was stable over multiple subcultures. Stable integration of the gfp gene into plant genomes was confirmed by Southern hybridization or polymerase chain reaction (PCR) analysis. We conclude that GFP can be used as a vital marker and reporter protein in transformation experiments with gymnosperms and deciduous trees.
