Evaluation of candidate genes expression in RNA interfering system (RNAi) in Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), vector of citrus huanglongbing

Orientador: Marcos Antonio MachadoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O psilídeo Diaphorina citri (Kuwayama) tornou-se nos últimos anos um dos mais importantes vetores na cultura de citros, em função dos crescentes prejuízos causados às plantas pela transmissão das bactérias Candidatus Liberibacter americanus e Candidatus Liberibacter asiaticus, agentes causais do huanglongbing (HLB) dos citros. O manejo do HLB é baseado no uso de mudas sadias, erradicação de plantas infectadas e pelo controle químico do vetor. No entanto, o uso contínuo e intenso de inseticidas é problemático, não só pelos custos financeiros, como também pelos problemas ambientais que o uso excessivo acarreta. Para reduzir os altos custos no desenvolvimento de novos inseticidas e tornar o controle mais eficiente e específico, o silenciamento gênico por RNA de interferência (RNAi) é uma das promessas de novas tecnologias utilizadas no controle de pragas e vetores. Assim, os objetivos deste trabalho foram buscar genes candidatos a serem silenciados, estabelecer sistemas de fornecimento de moléculas de dupla fita de RNA (dsRNAs), avaliar a mortalidade e expressão dos genes candidatos a silenciamento de D. citri. Para isso, foi utilizado um sistema de alimentação in vitro composto por diferentes concentrações de sacarose para avaliar a sobrevivência de D. citri nos estágios de ninfa e adultos durante cinco dias. Foi realizada a busca de 16 genes candidatos no transcriptoma e genoma de D. citri. Depois da confirmação das sequências, os genes inhibitor of apoptosis, trehalase, cathepsin D, cathepsin L, chitin synthase, Na+/K+ ATPase subunit-A e ATP synthase subunit-D like mitochondrial foram selecionados para serem transcritos in vitro e três diferentes concentrações de dsRNAs (200, 500 e 1000 ng.?L-1) de cada gene foram incorporadas em dieta artificial para insetos adultos. Também foi elaborado um sistema de fornecimento de dsRNAs de inhibitor of apoptosis, cathepsin D e chitin synthase via ramos de Murraya spp. para avaliar os efeitos de RNAi em ninfas. Nos ensaios de ingestão de dsRNAs via dieta artificial e ramos de murta foram avaliados a taxa de mortalidade e expressão gênica dos insetos, e também, a estabilidade dos dsRNAs. As concentrações 15 e 30% de sacarose no sistema de alimentação in vitro foram as que apresentaram maiores taxas de sobrevivência de D. citri adultas das populações estabelecidas no Centro de Citricultura `Sylvio Moreira¿/IAC e na Universidade da Califórnia-Davis, respectivamente. Após cinco dias, as taxas de mortalidade nos testes de fornecimento de dsRNAs dos genes alvos de D. citri em dieta artificial variaram entre 21-48%, 28-51% e 37-63%, nas concentrações 200, 500 e 1000 ng.?L-1, respectivamente. As taxas de sobrevivência de ninfas no sistema de fornecimento de dsRNAs de inhibitor of apoptosis, cathepsin D e chitin synthase via planta foram de 40, 71 e 50%, respectivamente. As moléculas de dsRNAs foram estáveis nos sistemas de fornecimento avaliados. Além disso, reduções nos níveis de expressão dos genes alvo de D. citri nos ensaios de RNAi foram confirmadas por PCR em tempo real. Estes dados sugerem métodos de fornecimento de dsRNAs, dieta artificial e ramos de murta, são ferramentas úteis para estudos de função gênica e possível controle de D. citriAbstract: In early years, the Asian citrus psyllid, Diaphorina citri (Kuwayama) has become one of the most important vectors in citrus, due to the increasing damage caused to plants by bacteria transmission, Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, causal agents of citrus huanglongbing (HLB). Healthy seedlings, eradication of infected plants and the chemical control of the vector have been used as HLB control strategies. However, the use constant and intensive of insecticides is not a good strategy, not only to the financial costs but also to the environmental problems. To reduce the high costs in development the new insecticides and making the control more efficient and specific, the gene knockdown by RNA interference (RNAi) is one of the promises of new technologies used in the control of pests and vectors. The aims of this study were to development double-stranded RNA (dsRNAs) delivery systems, searching candidate genes to be knockdown, evaluate the mortality and gene expression of D. citri. Thus, an in vitro feeding system composed of different sucrose concentrations was used to evaluate the nymph and adult D. citri survival for five days. After that, sixteen candidate genes were searching in transcriptome and genome D. citri. After confirmation of gene sequences, inhibitor of apoptosis, trehalase, cathepsin D, cathepsin L, chitin synthase, Na+/K+ ATPase subunit-A e ATP synthase subunit-D like mitochondrial genes were selected to be transcripts in vitro, and three different dsRNAs concentrations (200, 500 e 1000 ng.?L-1) of each gene were put in artificial diet to adult insects. Moreover, a dsRNAs (inhibitor of apoptosis, cathepsin D chitin synthase) delivery system by Murraya spp. leaves was development to evaluate the RNAi effects in nymphs. Mortality rates and gene expression of D. citri were evaluated after feeding dsRNAs via artificial diet and Murraya spp., and also, the dsRNAs stability in both delivery systems were evaluated. Fifteen and thirty percent sucrose in vitro feeding system showed the highest survival rates of adult D. citri populations from Citrus Center 'Sylvio Moreira' /IAC and University of California-Davis, respectively. After five days, the D. citri mortality rates in the dsRNAs delivery system of target genes on artificial diets ranged from 21-48%, 28-51% and 37-63% at concentrations of 200, 500 and 1000 ng.?L-1, respectively. Nymphs survival rates in the delivery system of inhibitor of apoptosis, cathepsin D and chitin synthase dsRNAs via plant leaves were 40, 71 and 50%, respectively. DsRNAs molecules were stable in the delivery systems evaluated. Furthermore, reduction in target gene expression levels of D. citri in the RNAi assays were confirmed by real time PCR. These datas suggest methods of providing dsRNAs, artificial diet and orange jasmine leaves, are useful tools for gene function studies and possible control of D. citriDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecular2013/02138-0FAPES

Management of pest insects and plant diseases by non-transformative RNAi

Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS - Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology

Oral Delivery Of Double-stranded Rnas Induces Mortality In Nymphs And Adults Of The Asian Citrus Psyllid, Diaphorina Citri

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.123Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [2013/02138-0, 2014/03925-8, 2008/57909-2]CNPq [573848/08-4]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Protection of coffee plants against brown eye spot by acibenzolar-S-methyl and harpin protein

O objetivo deste trabalho foi avaliar, em cafeeiro suscetível, a proteção contra a cercosporiose, pela aplicação da proteína harpina e acibenzolar-S-metil (ASM), e avaliar seu efeito na germinação de conídios e crescimento micelial in vitro. No primeiro experimento, cafeeiros tratados com ASM (25, 50, 100, 200 µg mL-1) receberam o inóculo de uma suspensão de conídios de Cercospora coffeicola, e a severidade da doença foi avaliada aos 30 e 60 dias após a inoculação. No segundo experimento, cafeeiros foram aspergidos com harpina (7,5, 15, 30, 60, 120 µg mL-1), tendo-se utilizado o mesmo procedimento. No terceiro experimento, plantas aspergidas previamente com ASM (200 µg mL-1) ou harpina (15 µg mL-1) foram tratadas novamente com esses produtos, aos 30 dias após terem recebido inóculo do patógeno. ASM e harpina protegeram os cafeeiros contra cercosporiose 30 dias após a inoculação com C. coffeicola. Entretanto, 60 dias após a inoculação, apenas o ASM (200 µg mL-1), com uma ou duas aplicações, protegeu as plantas contra C. coffeicola. Os cafeeiros foram protegidos contra cercosporiose, em reaplicação de harpina, 30 dias após o primeiro tratamento com essa proteína. Harpina e acibenzolar-S-metil não inibiram o desenvolvimento micelial nem a germinação in vitro dos conídios do patógeno.The objective of this work was to evaluate the protective effect of harpin protein and acibenzolar-S-methyl (ASM) against brown eye spot, in coffee plants, and its effect on in vitro conidial germination and mycelial growth of Cercospora coffeicola. In the first assay, plants treated with ASM (25, 50, 100, 200 µg mL-1) received the inoculum of a C. coffeicola conidial suspension, and the disease severity was evaluated 30 and 60 days after inoculation. In the second assay, plants were sprayed with harpin (7.5, 15, 30, 60, 120 µg mL-1) following the same procedure. In a third trial, plants previously sprayed with ASM (200 µg mL-1) or harpin (15 µg mL-1) were treated again with these products 30 days after pathogen inoculation. ASM and harpin protected the coffe plants against brown eye spot 30 days after inoculation with C. coffeicola. However, 60 days after inoculation, only ASM (200 µg mL-1), with one or two applications, conferred protection to plants against C. coffeicola. Coffee plants were protected against cercosporiosis, when harpin was reapplied on plants 30 days after a previous treatment with this protein. Harpin and acibenzolar-S-methyl did not inhibit the in vitro conidial germination and the mycelial growth of the pathogen

Genetic variability of Sugarcane mosaic virus causing maize mosaic in Brazil

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá‑los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS‑ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT‑PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.The objective of this work was to characterize biologically and molecularly three Sugarcane mosaic virus (SCMV) isolates from maize crops in Brazil, to analyze them phylogenetically and to discriminate genome polymorphisms. Plants with mosaic and stunting symptons were collected from maize crops in the state of São Paulo and in Rio Verde county, GO, Brazil; their foliar extracts were inoculated in indicator plants and subjected to serological analysis with antisera to SCMV, Maize dwarf mosaic virus (MDMV) and Johnsongrass mosaic virus (JGMV). Sorghum 'Rio' and 'TX 2786' seedlings showed mosaic symptons after inoculation of the three isolates, and DAS‑ELISA confirmed infection by SCMV in these plants. Total RNA was extracted from the infected leaves and was used as template for reverse transcriptase polymerase chain reaction (RT‑PCR). Specific fragments were amplified, subjected to restriction fragment length polymorphism (RFLP) analysis and sequenced. It was possible to discriminate the SCMV maize isolates from other Brazilian isolates of the virus. Multiple alignments and phylogenetic profile analyses corroborate these data and show diversity in the sequence of nucleotides that code for capsidal protein, what explains the distinct clustering of these isolates, suggesting that they should be ranked as distinct SCMV strains rather than geographical isolates

Genetic variability of Sugarcane mosaic virus causing maize mosaic in Brazil

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá‑los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS‑ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT‑PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.The objective of this work was to characterize biologically and molecularly three Sugarcane mosaic virus (SCMV) isolates from maize crops in Brazil, to analyze them phylogenetically and to discriminate genome polymorphisms. Plants with mosaic and stunting symptons were collected from maize crops in the state of São Paulo and in Rio Verde county, GO, Brazil; their foliar extracts were inoculated in indicator plants and subjected to serological analysis with antisera to SCMV, Maize dwarf mosaic virus (MDMV) and Johnsongrass mosaic virus (JGMV). Sorghum 'Rio' and 'TX 2786' seedlings showed mosaic symptons after inoculation of the three isolates, and DAS‑ELISA confirmed infection by SCMV in these plants. Total RNA was extracted from the infected leaves and was used as template for reverse transcriptase polymerase chain reaction (RT‑PCR). Specific fragments were amplified, subjected to restriction fragment length polymorphism (RFLP) analysis and sequenced. It was possible to discriminate the SCMV maize isolates from other Brazilian isolates of the virus. Multiple alignments and phylogenetic profile analyses corroborate these data and show diversity in the sequence of nucleotides that code for capsidal protein, what explains the distinct clustering of these isolates, suggesting that they should be ranked as distinct SCMV strains rather than geographical isolates

Development on infected citrus over generations increases vector infection by ‘candidatus liberibacter asiaticus in diaphorina citri’

‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus Huanglongbing (HLB), which is transmitted by Asian citrus psyllid (ACP), Diaphorina citri, causing severe losses in various regions of the world. Vector efficiency is higher when acquisition occurs by ACP immature stages and over longer feeding periods. In this context, our goal was to evaluate the progression of CLas population and infection rate over four ACP generations that continuously developed on infected citrus plants. We showed that the frequency of CLas-positive adult samples increased from 42% in the parental generation to 100% in the fourth generation developing on CLas-infected citrus. The bacterial population in the vector also increased over generations. This information reinforces the importance of HLB management strategies, such as vector control and eradication of diseased citrus trees, to avoid the development of CLas-infected ACP generations with higher bacterial loads and, likely, a higher probability of spreading the pathogen in citrus orchards118Insect Vectors of Plant PathogensCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ465440/2014-2; 310554/2016-02014/50880-0; 2015/13971-

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control