Alterações no perfil de metilação do DNA e identificação de transcritos diferencialmente expressos em cana-de-açúcar em resposta ao SCMV (Sugarcane mosaic virus)
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Data
2017-11-06
Autores
Silva, Marcel Fernando da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O mosaico da cana-de-açúcar é uma das principais viroses da cultura, sendo os estudos de padrões de metilação e de transcriptoma de contribuição para a compreensão da resistência genética à doença. O presente trabalho aplicou a técnica MSAP (“Methylation-sensitive amplified polymorphism”) em dois cultivares de cana-de-açúcar de resposta contrastante à doença do mosaico, causada pelo Potyvirus Sugarcane mosaic virus (SCMV), nas condições de inoculação falsa e inoculação com SCMV, para análise de alterações no padrão de metilação. As alterações no padrão de metilação causadas pela interação com o SCMV foram identificadas por meio de clonagem e sequenciamento. Fragmentos diferencialmente expressos (FDEs), previamente obtidos pela técnica cDNA-AFLP (“cDNA-amplified fragment length polymorphism”), também foram clonados e sequenciados para a identificação de genes candidatos associados à resistência ao SCMV. Em análises da frequência dos padrões de presença e ausência de bandas MSAP, níveis de metilação genômica variando de 33% a 35,4% foram observados para IAC91-1099, e de 33% a 37,3% para IACSP95-5000, com uma pequena proporção de loci alterados em resposta ao SCMV para ambos os cultivares. A análise global pelo pacote R MSAP indicou níveis de metilação genômica de 40%, além de demonstrar que as variações epigenéticas entre os cultivares (Phi_ST = 0,32; P = 0,008) apresentaram menor extensão que as variações genéticas (Phi_ST = 0,96; P = 0,0067). As análises AMOVA e PCoA confirmaram a pequena proporção de polimorfismos ocasionados pela infecção por SCMV, sendo as variações devidas a tempo de coleta mais proeminentes. Essas alterações, no entanto, foram específicas para os cultivares em estudo, sugerindo diferenças epigenéticas na resposta a inoculação com o SCMV. Enquanto o cultivar resistente IACSP95-5000 apresentou maiores polimorfismos de hipometilação 24 e 72 horas após a inoculação (hai) e a maior ocorrência de hipermetilação 48 hai, o cultivar suscetível IAC91-1099 apresentou uma troca entre a metilação da citosina interna e a semimetilação da citosina externa. O sequenciamento de polimorfismos MSAP decorrentes da interação com SCMV indicaram relevância para a caracterização da interação cana-de-açúcar e SCMV, uma vez que foram observados alinhamentos com transcritos com função putativa de resposta a estresses bióticos e abióticos, remodelação da cromatina e elementos transponíveis. Já os alinhamentos MSAP com região genômica revelaram possíveis regiões promotoras para os transcritos à jusante, relacionados à proteína kinase e elementos transponíveis. Os alinhamentos dos FDEs, oriundos do marcador molecular cDNA-AFLP, indicaram vias possivelmente relacionadas à fotossíntese, recuperação pós-estresse, proteínas transmembranas, resposta a estresses abióticos, elementos transponíveis e remodelação de cromatina via metilação de DNA e alterações na histona 3 (H3). A região promotora dos fragmentos MSAP e FDEs apresentou elementos reguladores responsivos a estresses, fitormônio e vias epigenéticas, além da ocorrência de ilhas CpG, sugerindo conexões entre alterações no transcriptoma e no perfil de metilação de DNA. A validação de três FDEs por qRT-PCR revelou uma complexidade na expressão e um comportamento diferente do observado por cDNA-AFLP. Ainda assim, o FDE_1 pode explicar alguns dos polimorfismos de hipometilação de DNA observados pelo marcador molecular MSAP, enquanto o FDE_2 pode explicar as diferenças na expressão de sintomas de mosaico entre os cultivares. Por sua vez, a regulação negativa do FDE_4 em IACSP95-5000 se assemelha ao observado na literatura para resistência genética em milho a um Potyvírus do subgrupo do SCMV.
Mosaic caused by Sugarcane mosaic virus (SCMV) is one of the main viruses infecting sugarcane worldwide. Studies on transcriptomic analysis and methylation status of genomic DNA contribute to understand the molecular bases of resistance to mosaic. The present study used the MSAP (Methylation-sensitive amplified polymorphism) approach in two sugarcane cultivars with contrasting response for SCMV, IACSP95-5000 (resistant) and IAC91-1099 (susceptible), both under mechanical inoculation condition, along with their respective mock inoculated controls, in order to evaluate changes in DNA methylation patterns and to identify polymorphisms caused by SCMV via cloning and sequencing. Differentially expressed transcribed fragments (DTFs) previously obtained from cDNA-AFLP were also cloned and sequenced with the aim of identifying candidate genes for SCMV resistance. Analysis of MSAP patterns frequency revealed genomic methylation levels ranging from 33% to 35.4% in IAC91-1099, and from 33% to 37.3% in IACSP95-5000, with minor changes caused by interaction with SCMV for both cultivars. Global analysis performed by R MSAP package demonstrates a genomic methylation level of 40% and also reveals that epigenetic variation between cultivars (Phi_ST = 0.32; P = 0.008) was less expressive than genetic variation (Phi_ST = 0.96; P = 0.0067). AMOVA and PCoA analyses confirmed the little extension of alterations in cytosine methylation caused by SCMV inoculation, being the variations observed among sample time points more expressive. Nonetheless, SCMV changes in cytosine methylation were distinctive between the cultivars, suggesting epigenetic differences in response to SCMV inoculation. Whilst the resistant cultivar IACSP95- 5000 showed higher frequencies of polymorphisms regarding cytosine hyphomethylation 24 hours post inoculation (hpi) and 72 hpi, and higher hypermethylation polymorphisms 48 hpi, the susceptible cultivar IACSP91-1099 showed an exchange from inner cytosine methylation to external cytosine hemimethylation. The sequencing of the MSAP polymorphism caused by SCMV indicated relevance for the characterization of sugarcane-SCMV interaction, since alignments with transcripts putatively associated with biotic and abiotic stresses responses, chromatin remodeling and transposable elements were observed. In turn, MSAP alignments with genomic region showed putative promoter regions for downstream transcripts associated with protein kinase and transposable elements. DTFs alignments suggests changes in photosynthesis, post-stress recovery, activity of transmembrane proteins, responses to biotic and abiotic stresses, transposable elements and a DTF with putative function in chromatin remodeling via DNA methylation and histone 3 (H3) modification. Analyses of putative promoter regions of MSAP fragments and DTFs revealed cis acting motifs responsive to stress, phytohormones and epigenetic pathways, besides the proximity of CpG islands, suggesting a crosstalk between changes in transcriptome and cytosine methylation. The validation of three DTFs by qRT-PCR showed a complex expression and a contrasting behavior from cDNA-AFLP observations. Even though, DTF_1 profile expression may explain some of the hypomethylation polymorphisms observed from MSAP molecular marker, whereas DTF_2 may explain the contrasting behavior in symptom expression between cultivars. The pronounced downregulation observed for DTF_4 in IACSP95-5000, in turn, correlates with maize genetic resistance to a Potyvirus belonging to SCMV subgroup.
Mosaic caused by Sugarcane mosaic virus (SCMV) is one of the main viruses infecting sugarcane worldwide. Studies on transcriptomic analysis and methylation status of genomic DNA contribute to understand the molecular bases of resistance to mosaic. The present study used the MSAP (Methylation-sensitive amplified polymorphism) approach in two sugarcane cultivars with contrasting response for SCMV, IACSP95-5000 (resistant) and IAC91-1099 (susceptible), both under mechanical inoculation condition, along with their respective mock inoculated controls, in order to evaluate changes in DNA methylation patterns and to identify polymorphisms caused by SCMV via cloning and sequencing. Differentially expressed transcribed fragments (DTFs) previously obtained from cDNA-AFLP were also cloned and sequenced with the aim of identifying candidate genes for SCMV resistance. Analysis of MSAP patterns frequency revealed genomic methylation levels ranging from 33% to 35.4% in IAC91-1099, and from 33% to 37.3% in IACSP95-5000, with minor changes caused by interaction with SCMV for both cultivars. Global analysis performed by R MSAP package demonstrates a genomic methylation level of 40% and also reveals that epigenetic variation between cultivars (Phi_ST = 0.32; P = 0.008) was less expressive than genetic variation (Phi_ST = 0.96; P = 0.0067). AMOVA and PCoA analyses confirmed the little extension of alterations in cytosine methylation caused by SCMV inoculation, being the variations observed among sample time points more expressive. Nonetheless, SCMV changes in cytosine methylation were distinctive between the cultivars, suggesting epigenetic differences in response to SCMV inoculation. Whilst the resistant cultivar IACSP95- 5000 showed higher frequencies of polymorphisms regarding cytosine hyphomethylation 24 hours post inoculation (hpi) and 72 hpi, and higher hypermethylation polymorphisms 48 hpi, the susceptible cultivar IACSP91-1099 showed an exchange from inner cytosine methylation to external cytosine hemimethylation. The sequencing of the MSAP polymorphism caused by SCMV indicated relevance for the characterization of sugarcane-SCMV interaction, since alignments with transcripts putatively associated with biotic and abiotic stresses responses, chromatin remodeling and transposable elements were observed. In turn, MSAP alignments with genomic region showed putative promoter regions for downstream transcripts associated with protein kinase and transposable elements. DTFs alignments suggests changes in photosynthesis, post-stress recovery, activity of transmembrane proteins, responses to biotic and abiotic stresses, transposable elements and a DTF with putative function in chromatin remodeling via DNA methylation and histone 3 (H3) modification. Analyses of putative promoter regions of MSAP fragments and DTFs revealed cis acting motifs responsive to stress, phytohormones and epigenetic pathways, besides the proximity of CpG islands, suggesting a crosstalk between changes in transcriptome and cytosine methylation. The validation of three DTFs by qRT-PCR showed a complex expression and a contrasting behavior from cDNA-AFLP observations. Even though, DTF_1 profile expression may explain some of the hypomethylation polymorphisms observed from MSAP molecular marker, whereas DTF_2 may explain the contrasting behavior in symptom expression between cultivars. The pronounced downregulation observed for DTF_4 in IACSP95-5000, in turn, correlates with maize genetic resistance to a Potyvirus belonging to SCMV subgroup.
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Palavras-chave
epigenética, genes candidatos, infecção viral, perfil de expressão, Saccharum officinarum, epigenetics, candidate genes, viral infeccion, expression profile