Análise funcional das ORFs XAC2361 e XAC3898 de Xanthomonas citri subsp. citri agente causal do cancro cítrico
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2018-03-02
Autores
Kempner, Tamiris
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Universidade Estadual Paulista (Unesp)
Resumo
Diante da importância do cancro cítrico para a citricultura mundial, a bactéria Xanthomonas citri subsp. citri, o agente causal da doença, tem sido amplamente estudada. Pesquisadores brasileiros realizaram o sequenciamento completo do genoma da X. citri subsp. citri isolado 306 (Xac306), com o intuito de elucidar os genes e mecanismos envolvidos na patogenicidade e/ou virulência da bactéria. As ORFs XAC2361 e XAC3898 possuem tamanhos e locais diferentes no genoma da Xac306, mas têm em comum o domínio Peptidase_M23, cujo processo biológico predito é de hidrolase de parede celular, mas a real função destas proteínas em Xac ainda é desconhecida. Este trabalho teve como objetivo avaliar, por meio da mutação sítio-dirigida, se as ORFs XAC2361 e XAC3898 estão relacionadas com a virulência e/ou patogenicidade de X. citri subsp. citri 306 e com o desenvolvimento do cancro cítrico. Para isso, foram realizados testes in vitro e in planta dos mutantes obtidos, visando observar alterações fenotípicas. Devido a presença de peptídeo sinal, a ORF XAC2361 foi fundida à proteína fluorescente mCherry (XAC2361-mCherry) para expressão in vivo e avaliação por microscopia de fluorescência. As análises fenotípicas demonstraram que o mutante ΔXAC2361 não apresentou alterações nos sintomas quando avaliado em limoeiro cravo (Citrus limonia Osbeck), porém apresentou menor capacidade de crescimento in vitro, menor motilidade swimming e swarming e menor capacidade de sobrevivência em SDS. Por outro lado, o mutante ΔXAC3898 apresentou uma significativa redução na virulência, menor capacidade de crescimento in planta, maior capacidade de crescimento in vitro, menor capacidade de agregação, maior produção de goma e biofilme e a avaliação por microscopia eletrônica de varredura mostrou que houve uma redução no comprimento das células da bactéria. A análise por microscopia de florescência da proteína de fusão XAC2361-mCherry mostrou, predominantemente, uma fluorescência dispersa, o que é um indício de que a mesma pode estar sendo direcionada para o citoplasma. Apesar disso, não foi possível confirmar a expressão da proteína de fusão XAC2361-mCherry por meio de Western-blot. Diante dos resultados obtidos, sugere-se que o domínio Peptidase_M23 está de alguma forma envolvido com a virulência e/ou patogenicidade de Xac, atuando na formação da parede celular.
In view of the importance of citrus canker in citrus culture worlwide, the bacterium Xanthomonas citri subsp. citri, the causal agent of this disease, has been extensively studied. Brazilian researchers performed the complete sequencing of the genome of X. citri subsp. citri isolate 306 (Xac306), in order to elucidate the genes and mechanisms involved in the pathogenicity and/or virulence of Xac. The ORFs XAC2361 and XAC3898 have different sizes and location in the genome, but have in common the M23 Peptidase domain, whose biological process is predicted to act as cell wall hydrolase, but the actual function of these proteins in Xac is still unknown. This work aimed to evaluate, through site-directed mutagenesis, whether XAC2361 and XAC3898 ORFs are related to the virulence and / or pathogenicity of Xac306 and the development of citrus canker. In order to observe phenotypic changes, in vitro and in planta tests of the mutants were carried out. Due to the presence of signal peptide, the ORF XAC2361 was fused to the mCherry fluorescent protein (XAC2361-mCherry) for in vivo expression and fluorescence microscopy evaluation. Phenotypic analyzes demonstrated that the ΔXAC2361 mutant showed no change in symptoms when evaluated in Mexican lime (Citrus limonia Osbeck), but presented lower in vitro growth capacity, lower swimming and swarming motility, and lower survival capacity in SDS. On the other hand, the ΔXAC3898 mutant showed a significant reduction in virulence, lower in plant growth capacity, higher in vitro growth capacity, lower aggregation capacity, higher gum production and biofilm, and scanning electron microscopy evaluation showed that there was a reduction in cell length. Microscopy analysis of the XAC2361-mCherry fusion protein showed predominantly dispersed fluorescence, which is an indication that it may be being directed to the cytoplasm. Despite this, the expression of XAC2361-mCherry fusion protein could not be confirmed by Western blotting. In view of the obtained results, it is suggested that the Peptidase_M23 domain is in some way involved with the virulence and/or pathogenicity of Xac, acting in the formation of the cellular wall.
In view of the importance of citrus canker in citrus culture worlwide, the bacterium Xanthomonas citri subsp. citri, the causal agent of this disease, has been extensively studied. Brazilian researchers performed the complete sequencing of the genome of X. citri subsp. citri isolate 306 (Xac306), in order to elucidate the genes and mechanisms involved in the pathogenicity and/or virulence of Xac. The ORFs XAC2361 and XAC3898 have different sizes and location in the genome, but have in common the M23 Peptidase domain, whose biological process is predicted to act as cell wall hydrolase, but the actual function of these proteins in Xac is still unknown. This work aimed to evaluate, through site-directed mutagenesis, whether XAC2361 and XAC3898 ORFs are related to the virulence and / or pathogenicity of Xac306 and the development of citrus canker. In order to observe phenotypic changes, in vitro and in planta tests of the mutants were carried out. Due to the presence of signal peptide, the ORF XAC2361 was fused to the mCherry fluorescent protein (XAC2361-mCherry) for in vivo expression and fluorescence microscopy evaluation. Phenotypic analyzes demonstrated that the ΔXAC2361 mutant showed no change in symptoms when evaluated in Mexican lime (Citrus limonia Osbeck), but presented lower in vitro growth capacity, lower swimming and swarming motility, and lower survival capacity in SDS. On the other hand, the ΔXAC3898 mutant showed a significant reduction in virulence, lower in plant growth capacity, higher in vitro growth capacity, lower aggregation capacity, higher gum production and biofilm, and scanning electron microscopy evaluation showed that there was a reduction in cell length. Microscopy analysis of the XAC2361-mCherry fusion protein showed predominantly dispersed fluorescence, which is an indication that it may be being directed to the cytoplasm. Despite this, the expression of XAC2361-mCherry fusion protein could not be confirmed by Western blotting. In view of the obtained results, it is suggested that the Peptidase_M23 domain is in some way involved with the virulence and/or pathogenicity of Xac, acting in the formation of the cellular wall.
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Palavras-chave
Microscopia, Mutagênese sítio-dirigida, Patogenicidade, Peptidase-M23, Virulência