Transcritômica e proteômica aplicadas à prospecção de genes candidatos envolvidos na resposta de defesa aos estresses biótico e abiótico em Arachis spp.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2019.Muitos fatores bióticos e abióticos podem limitar a produtividade e a qualidade dos grãos de amendoim, especialmente considerando que essa cultura é amplamente cultivada nos trópicos semiáridos e por agricultores com poucos recursos. Tanto a seca quanto os nematoides das galhas (Meloidogyne spp.) são problemas que afetam não apenas a cultura do amendoim, mas também outras culturas, representando uma ameaça mundial à produção agrícola e a segurança alimentar. O amendoim (Arachis hypogaea) apresenta alta suscetibilidade a diferentes estresses ambientais e ataques de patógenos. Por outro lado, seus parentes silvestres apresentam maior adaptabilidade a ambientes adversos e resistência a inúmeros fitopatógenos, mostrando-se importantes fontes de resistência alélica aos estresses bióticos e abióticos para o melhoramento genético do amendoim. Neste estudo, proteínas obtidas a partir de tecidos radiculares tanto de plantas de A. duranensis submetidas a diminuição gradual de água no solo quanto de plantas de A. stenosperma inoculadas com nematoide das galhas, foram analisadas, assim como sua abundância diferencial em relação aos perfis de expressão de seus transcritos correspondentes obtidos por RNA-Seq e RT-qPCR. Utilizando a abordagem 2-DE, um total de 31 proteínas diferencialmente abundantes (DAPs) foram identificadas em raízes de A. duranensis e 21 em raízes de A. stenosperma. Além disso, 222 DAPs também foram identificadas pela análise 2D-NanoUPLC-MSE em raízes de A. stenosperma. A correlação entre a abundância proteica e a expressão do mRNA correspondente (in silico e in vitro) mostrou que enquanto a dinâmica de resposta ao estresse hídrico foi preferencialmente negativamente regulada em raízes estressadas de A. duranensis, em raízes de A. stenosperma infectadas com nematoide foi positivamente regulada. Entre as proteínas aqui identificadas, duas proteínas relacionadas a patogênese (PRs), ambas responsivas à seca e uma delas responsiva também ao ataque do nematoide, foram selecionadas para avaliação quanto ao efeito de sua superexpressão em resposta a esses estresses. Este estudo demonstra a utilidade de estudos proteômicos e sua correlação com dados de transcrição, na identificação de genes e reguladores envolvidos na resposta de defesa ao estresse biótico e abiótico em espécies com relativamente poucos recursos genômicos como o Arachis. As proteínas identificadas são potencialmente úteis para o melhoramento genético do amendoim cultivado visando a tolerância à seca e a resistência ao ataque de patógenos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Pesquisas (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Many biotic and abiotic factors may limit the productivity and quality of peanut grains, especially considering that this crop is widely cultivated in the semi-arid tropics and by poor farmers. Both drought and root-knot nematodes (RKN – Meloidogyne spp.) are problems that affect not only the peanut crop, but also other crops, posing a worldwide threat to agricultural production and food security. Peanut (Arachis hypogaea) presents high susceptibility to different environmental stresses and pathogen attacks. On the other hand, its wild relatives show greater adaptability to adverse environments and resistance to numerous phytopathogens, proving to be important sources of resistance alleles to biotic and abiotic stresses for the genetic improvement of peanuts. In this study, proteins obtained from root tissues of both A. duranensis plants subjected to the gradual reduction of water in the soil and from A. stenosperma plants inoculated with RKN were analyzed, as well as their differential abundance in relation to the profiles of their corresponding transcripts obtained by RNA-seq and RT-qPCR. Using the 2-DE approach, a total of 31 differentially abundant proteins (DAPs) were identified in roots of A. duranensis and 21 in roots of A. stenosperma. In addition, 222 DAPs were also identified by 2D-NanoUPLC-MSE analysis on A. stenosperma roots. The correlation between protein abundance and the expression of the corresponding mRNA (in silico and in vitro) showed that while the dynamics of response to water deficit was preferentially negatively regulated in stressed roots of A. duranensis, roots of A. stenosperma infected with nematoid was positively regulated. Among the proteins identified herein, two pathogenesis-related proteins (PRs), both responsive to drought and one of them also responsive to nematode attack, were selected for evaluation as to the effect of their overexpression in response to these stresses. In addition, this study demonstrates the usefulness of proteomic studies and their correlation with transcription data, in the identification of genes and regulators involved in the defense response to biotic and abiotic stress in species with relatively few genomic resources such as Arachis. The identified proteins are potentially useful for the genetic improvement of cultivated peanuts aiming at drought tolerance and resistance to pathogen attack

Prospecção e validação de genes-candidatos envolvidos na resistência de amendoim silvestre (Arachis stenosperma) ao Passalora personata, o agente causal da mancha preta do amendoim

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Pós-Graduação em Fitopatologia, 2011.O amendoim cultivado (Arachis hypogaea) é umas das oleaginosas mais importantes no mundo, pelo seu grande valor energético e nutricional. Contudo, o amendoim possui baixa variabilidade genética quando consideradas características de interesse agronômico, o que difere de suas espécies silvestres, as quais são fontes de resistência a doenças e de adaptação a diversos ambientes. Arachis stenosperma é uma espécie silvestre resistente a algumas pragas e doenças, dentre elas, a mancha preta do amendoim, causada pelo fungo Passalora personata. Visando a identificação de genescandidatos associados à resposta de defesa da planta, o transcritoma de plantas de A. stenosperma acesso V10309, desafiadas com o fungo P. personata, e seu controle, foram analisados através de pirossequencimento massal (Roche), resultando em 362.631 genes transcritos, o que gerou 17.912 unigenes, dos quais 7.723 eram constituídos por contigs. Dentre as anotações obtidas por BLASTx, 50 fatores de transcrição, 48 RGAs e vários genes-candidatos envolvidos em processos de defesa da planta foram identificados. Dentre eles, 16 genes-candidatos, cinco deles RGAs, foram avaliados através de RT-qPCR, quanto ao nível de expressão mediante a presença do patógeno, sendo cinco dos 11 primeiros, negativamente regulados, enquanto que todos os RGAs foram positivamente regulados. Esta é a primeira análise massal do transcritoma de espécie silvestre de Arachis inoculado com P. personata. Os dados gerados neste trabalho promoverão o enriquecimento do banco de dados de genes transcritos de A. stenosperma, os quais poderão ser utilizados para descoberta de genes-candidatos à resistência, a outras características de interesse agronômico e novos marcadores moleculares (SNPs e SSRs), que uma vez mapeados, poderão auxiliar na identificação de QTLs no melhoramento da cultura do amendoim, constituindo um importante recurso para a comunidade científica interessada na descoberta de genes relacionados à resposta de defesa ou ao estresse biótico em amendoim e em outras espécies. _________________________________________________________________________________ ABSTRACTPeanut (Arachis hypogaea) is one of the most important oilseeds in the world due to its high energy value and nutrition. However, peanut has a low genetic variability, especially for agronomical traits, which differs from its wild species, which are sources of disease resistance and adaptation to various environments. Arachis stenosperma is a wild species resistant to some pests and diseases, among them, the Late Leaf Spot, caused by the fungus Passalora personata. Aiming to identify candidate genes associated with the plant defense response, the transcriptome of A. stenosperma V10309, challenged with the fungus P. personata and from a non-infected control, was analyzed by large scale pyrosequencing (Roche), resulting in 362,631 expressed genes, which generated 17,912 unigenes, which consisted of 7,723 contigs. Among the annotations obtained by BLASTx, 50 transcription factors, 48 RGAs and several candidate genes involved in plant defense processes were identified. Among them, 16 candidate genes were analyzed through RTqPCR for expression in the presence of the pathogen. Five RGAs were up-regulated, whilst the other 11 were down-regulated. This is the first large scale report on the transcriptome analysis of wild peanut inoculated with P. personata. The data generated in this study promote the enrichment of the database of expressed genes from A. stenosperma, which can be used for discovery of candidate genes resistance, other agronomic traits of interest and new molecular markers (SNPs and SSRs). Once mapped, these can assist in the identification of QTLs in peanut crop improvement. This data is an important resource to the scientific community in the discovery of genes related to defense response or biotic stress in peanuts and other species

Transcriptome Responses of Wild Arachis to UV-C Exposure Reveal Genes Involved in General Plant Defense and Priming

Stress priming is an important strategy for enhancing plant defense capacity to deal with environmental challenges and involves reprogrammed transcriptional responses. Although ultraviolet (UV) light exposure is a widely adopted approach to elicit stress memory and tolerance in plants, the molecular mechanisms underlying UV-mediated plant priming tolerance are not fully understood. Here, we investigated the changes in the global transcriptome profile of wild Arachis stenosperma leaves in response to UV-C exposure. A total of 5751 differentially expressed genes (DEGs) were identified, with the majority associated with cell signaling, protein dynamics, hormonal and transcriptional regulation, and secondary metabolic pathways. The expression profiles of DEGs known as indicators of priming state, such as transcription factors, transcriptional regulators and protein kinases, were further characterized. A meta-analysis, followed by qRT-PCR validation, identified 18 metaDEGs as being commonly regulated in response to UV and other primary stresses. These genes are involved in secondary metabolism, basal immunity, cell wall structure and integrity, and may constitute important players in the general defense processes and establishment of a priming state in A. stenosperma. Our findings contribute to a better understanding of transcriptional dynamics involved in wild Arachis adaptation to stressful conditions of their natural habitats

Engineering Resistance against <i>Sclerotinia sclerotiorum</i> Using a Truncated NLR (TNx) and a Defense-Priming Gene

The association of both cell-surface PRRs (Pattern Recognition Receptors) and intracellular receptor NLRs (Nucleotide-Binding Leucine-Rich Repeat) in engineered plants have the potential to activate strong defenses against a broad range of pathogens. Here, we describe the identification, characterization, and in planta functional analysis of a novel truncated NLR (TNx) gene from the wild species Arachis stenosperma (AsTIR19), with a protein structure lacking the C-terminal LRR (Leucine Rich Repeat) domain involved in pathogen perception. Overexpression of AsTIR19 in tobacco plants led to a significant reduction in infection caused by Sclerotinia sclerotiorum, with a further reduction in pyramid lines containing an expansin-like B gene (AdEXLB8) potentially involved in defense priming. Transcription analysis of tobacco transgenic lines revealed induction of hormone defense pathways (SA; JA-ET) and PRs (Pathogenesis-Related proteins) production. The strong upregulation of the respiratory burst oxidase homolog D (RbohD) gene in the pyramid lines suggests its central role in mediating immune responses in plants co-expressing the two transgenes, with reactive oxygen species (ROS) production enhanced by AdEXLB8 cues leading to stronger defense response. Here, we demonstrate that the association of potential priming elicitors and truncated NLRs can produce a synergistic effect on fungal resistance, constituting a promising strategy for improved, non-specific resistance to plant pathogens

A novel soybean hairy root system for gene functional validation.

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean

Defining the combined stress response in wild Arachis

International audienceNematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis . Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene ( AsECHI ) from Arachis stenosperma , reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance