Insights on the interaction between Rutaceae genotypes and the ‘Candidatus liberibacter asiaticus’ bacterium
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2022-12-20
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Universidade Estadual Paulista (Unesp)
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No Brasil, a doença Huanglongbing (HLB) dos citros está associada principalmente à presença da bactéria floemática ‘Candidatus Liberibacter asiaticus’ (Las), a qual é transmitida pelo inseto vetor Diaphorina citri. Embora todas as espécies de Citrus sejam suscetíveis, Microcitrus e Eremocitrus resistentes a Las foram recentemente relatados. Com o objetivo de obter mais informações sobre o fenótipo resistente, um conjunto de cinco experimentos foi feito por meio dos quais avaliamos: (1) aquisição de Las por D. citri alimentados em genótipos resistentes; (2) Infecção de Las em plantas de laranjeira doce enxertadas com casca ou borbulhas dos materiais resistentes; (3) Infecção por Las em plantas de laranjeira doce enxertadas sobre os materiais resistentes; (4) infecção de Las em novos brotos de plantas enraizadas nos materiais resistentes; e (5) Infecção de Las em novos brotos após poda drástica. Os resultados confirmaram a resistência total desses materiais e uma análise morfológica e de genotipagem por sequenciamento confirmou as informações genéticas essenciais para mapear adequadamente regiões genômicas para um programa de melhoramento de citros. Além desses genótipos, Murraya paniculata e Bergera koeniggi foram descritos como hospedeiros transitórios e imunes à bactéria HLB, respectivamente. No entanto, diferentemente de Eremocitrus e Microcitrus, não são compatíveis para enxertia e nem cruzadas com Citrus. Com o objetivo de obter mais informações sobre a interação Las-planta nessas duas espécies e na laranja doce, estudamos a multiplicação de Las imediatamente após a inoculação por psilídeos infectados alimentando-se deles por 2 dias. Observamos que logo após a remoção dos psilídeos, os títulos de Las caíram até o 12º dia nas três espécies. Depois disso, Las aumentou exponencialmente em C. × sinensis e M. paniculata. Em C. × sinensis, Las atingiu uma fase estacionária a partir do 40º dia, enquanto em M. paniculata, o título de Las aumentou a uma taxa menor entre o 40º e 60º dias, diminuindo gradativamente a partir daí. Em B. koenigii, o título diminuiu desde o início e permaneceu indetectável ao longo do experimento. Além disso, observamos que foram necessários 23,6 dias para Las se deslocar do fluxo onde foi feita a inoculação e colonizar toda a planta de C. × sinensis. Após essas observações, propusemos uma análise de RNA-seq nos pontos de tempo-chave que selecionamos como mais cruciais para respostas de resistência ou suscetibilidade. Ao comparar plantas expostas a insetos Las+ vs insetos saudáveis, observou-se um baixo número de genes significativamente expressos de forma diferente. No entanto, ao comparar amostras inoculadas com amostras não expostas a insetos, observou-se que B. koenigii não sofreu nenhuma alteração na expressão e interrompeu amplamente seu transcriptoma devido à infecção até 10 dias após a exposição aos insetos Las+. A análise de GSEA sugeriu que a fotossíntese foi restrita quando as amostras foram expostas a insetos Las+, sendo que estes decrescem mais forte em B. koenigii do que em laranja doce. Em conjunto, nossos resultados permitiram observar que a resposta de defesa das células vegetais às infecções por Las só foi ativada após alguns dias de infecção bacteriana. No entanto, mais análises devem ser feitas para tentar obter mais informações sobre essas interações.
In Brazil, the Citrus disease Huanglongbing (HLB) is mostly associated with the presence of the phloem-limiting and unculturable ‘Candidatus Liberibacter asiaticus’ which is transmitted by the insect vector Diaphorina citri. Although all Citrus species are susceptible, Las resistant Microcitrus and Eremocitrus have recently been reported. Aiming to gain more insights on the resistant phenotype, a set of five experiments were done through which we evaluated: (1) Las acquisition by D. citri fed onto the resistant genotypes; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back pruning. Results confirmed the full-resistance of those materials and a morphological and genotyping by sequencing analysis confirmed the genetic background informations that are essential to map properly genomic regions for a citrus breeding program. Apar from those genotypes, Murraya paniculata and Bergera koeniggi have been described as transient and immune hosts to the HLB bacterium, respectively. However, differently from Eremocitrus and Microcitrus, they are neither graft nor cross-compatible with Citrus. Aiming to gain more information of the Las-plant host interaction on these two species and sweet orange, we studied the time course of Las multiplication and whole-plant colonization immediately after the inoculation by infected psyllids feeding onto them for 2 days. We observed that soon after psyllids removal, Las titers dropped until the 12th days in all three species. After that, Las increased exponentially in C. × sinensis and M. paniculata. In C. × sinensis, Las reached a stationary phase from the 40th day onward, while in M. paniculata, Las titer increased at a lower rate between the 40th and 60th days, decreasing gradually thereafter. In B. koenigii, Las titer decreased from the start and remained undetectable along the experiment. Furthermore, we observed that it was required 23.6 days for Las to move from the flush where inoculation was done and colonize the entire plant of C. × sinensis. Following those observations, we proposed an RNA-seq analysis on the key-time points that we selected as most crucial for either resistance or susceptibility responses. When comparing plants exposed to Las+ insects vs healthy insects a low number of significant differently expressed genes were observed. However, when comparing challenged-inoculated samples with samples not exposed to insects it was observed that B. koenigii did not undergone any change in expression and largely arrested their transcriptome due to infection until 10 days after being exposed to Las+ insects. GSEA analysis suggested that photosynthesis was restricted when samples were exposed to Las+ insects, being these decress stronger in B. koenigii than in sweet orange. Taken together, our results allowed us to observe that plant cell defense response to Las infections was only active after few days of bacteral infection. However, more analysis should be done to try to gain more insight on those interactions.
In Brazil, the Citrus disease Huanglongbing (HLB) is mostly associated with the presence of the phloem-limiting and unculturable ‘Candidatus Liberibacter asiaticus’ which is transmitted by the insect vector Diaphorina citri. Although all Citrus species are susceptible, Las resistant Microcitrus and Eremocitrus have recently been reported. Aiming to gain more insights on the resistant phenotype, a set of five experiments were done through which we evaluated: (1) Las acquisition by D. citri fed onto the resistant genotypes; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back pruning. Results confirmed the full-resistance of those materials and a morphological and genotyping by sequencing analysis confirmed the genetic background informations that are essential to map properly genomic regions for a citrus breeding program. Apar from those genotypes, Murraya paniculata and Bergera koeniggi have been described as transient and immune hosts to the HLB bacterium, respectively. However, differently from Eremocitrus and Microcitrus, they are neither graft nor cross-compatible with Citrus. Aiming to gain more information of the Las-plant host interaction on these two species and sweet orange, we studied the time course of Las multiplication and whole-plant colonization immediately after the inoculation by infected psyllids feeding onto them for 2 days. We observed that soon after psyllids removal, Las titers dropped until the 12th days in all three species. After that, Las increased exponentially in C. × sinensis and M. paniculata. In C. × sinensis, Las reached a stationary phase from the 40th day onward, while in M. paniculata, Las titer increased at a lower rate between the 40th and 60th days, decreasing gradually thereafter. In B. koenigii, Las titer decreased from the start and remained undetectable along the experiment. Furthermore, we observed that it was required 23.6 days for Las to move from the flush where inoculation was done and colonize the entire plant of C. × sinensis. Following those observations, we proposed an RNA-seq analysis on the key-time points that we selected as most crucial for either resistance or susceptibility responses. When comparing plants exposed to Las+ insects vs healthy insects a low number of significant differently expressed genes were observed. However, when comparing challenged-inoculated samples with samples not exposed to insects it was observed that B. koenigii did not undergone any change in expression and largely arrested their transcriptome due to infection until 10 days after being exposed to Las+ insects. GSEA analysis suggested that photosynthesis was restricted when samples were exposed to Las+ insects, being these decress stronger in B. koenigii than in sweet orange. Taken together, our results allowed us to observe that plant cell defense response to Las infections was only active after few days of bacteral infection. However, more analysis should be done to try to gain more insight on those interactions.
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Citricos, Melhoramento genético, Biotecnologia Vegetal, Fitopatologia, citrus breeding, greening, HLB