Selection of reference genes for expression Study in pulp and seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality—selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality. (Résumé d'auteur

Genômica funcional da interação cupuaçuzeiro (Theobroma grandiflorum) e Moniliophthora perniciosa, agente causal da vassoura de bruxa

Tese (doutorado) — Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2023.O Theobroma grandiflorum é uma fruteira nativa da região amazônica e parente do cacau (T. cacao). Possui um enorme potencial econômico devido aos seus múltiplos usos nas indústrias alimentícia e cosmética. A cultura do cupuaçu é gravemente afetada pelo Moniliophthora perniciosa, fungo causador da vassoura de bruxa (VB), tanto no cupuaçu quanto no cacau. O conhecimento desse fitopatossistema é fundamental para propor estratégias de controle e mitigação dos danos causados pela VB a essas culturas. Para fornecer informações sobre a resistência do cupuaçu a M. perniciosa, os perfis transcritômicos de um genótipo resistente (clone 174) e um suscetível (clone 1074) foram analisados usando a tecnologia RNA-seq. Neste estudo, apresentamos a análise do transcritoma de ambos os genótipos desafiados com M. perniciosa, em diferentes tempos de exposição ao patógeno, na fase inicial da infecção. Um total de 21.441 unigenes e 440 genes diferencialmente expressos (DEGs) foram identificados entre as diferentes condições. A análise de diferença intrínseca entre os genótipos mostrou 301 DEGs. A alteração da expressão gênica foi observada mais precocemente no genótipo suscetível 24 horas após a inoculação (hai). No genótipo resistente, a alteração foi mais acentuada às 48 hai. Este conjunto de dados permitiu a identificação de genes potencialmente envolvidos nos mecanismos de defesa, entre eles: receptores de reconhecimento de padrões (PRRs); fatores de transcrição; proteínas relacionadas à patogênese (PRs); proteínas relacionadas ao remodelamento da parede celular; genes relacionados ao acúmulo de espécies reativas de oxigênio (ROS) e vias de terpenos. A análise da assinatura dos fitohormônios, em cada condição, revelou uma influência hormonal significativa nas respostas dos genótipos. Os genótipos diferiram principalmente em relação às respostas de auxina, citocinina, ácido salicílico e brassinosteróides. Este é o primeiro estudo de transcriptoma em larga escala de T. grandiflorum, que além de conhecimento sobre o processo de resistência, gerou uma lista de genes potencialmente importantes neste processo. Três genes desta lista foram selecionados para avaliação funcional por expressão heteróloga em tomate Micro-Tom (MT): 1) TgERF9 que codifica para um fator de transcrição, 2) TgTPL1 que codifica para uma proteína semelhante a taumatina e 3) TgPR10.1. As plantas transformadas, expressando o gene de interesse, foram desafiadas com fungos fitopatogênicos hemibiotróficos (Fusarium oxysporum f. sp. lycopersici raça 3 e Verticillium dahliae raça 2 e um fungo necrotrófico (Sclerotinia sclerotiorum). As plantas MT_TgERF9 e MT_TgPR10.1 foram desafiadas com M. perniciosa. Além disso, foi realizado um estudo da localização de transcritos de TgPR10.1 nos tecidos da gema apical de cupuaçuzeiro, via hibridização in situ (ISH). Todos os patógenos foram capazes de colonizar os tecidos das plantas transformadas e não transformadas. Contudo, apesar de ser visível o escurecimento interno do caule nas plantas inoculadas com Verticillium e Fusarium, não foram observados sintomas de murcha, mesmo em situação de déficit hídrico. Além disso, o crescimento das plantas e a produção de frutos não foram alterados em função da infecção. No entanto, as plantas transformadas com TgERF9 e TgTLP1 apresentaram uma tendência a serem menores e menos produtivas. Os bioensaios com folhas destacadas indicam que a expressão de TgTLP1 aumentou a suscetibilidade de MT ao fungo S. sclerotiorum. Plantas MT_TgPR10.1 apresentaram resistência moderada a S. sclerotiorum. A expressão deste gene em MT não afetou o desenvolvimento da VB. Entretanto, MT_TgPR10.1 apresentou alteração na altura, indicando alteração de balanço hormonal. No que se refere a localização dos transcritos de TgPR10.1 em cupuaçu, foi possível identificar a expressão deste gene nos tricomas, no procâmbio, no meristema e nas células da epiderme dos primórdios foliares. A presença destes transcritos, particularmente no procâmbio, indica um papel desta PR no desenvolvimento e crescimento da planta, os quais são afetados por citocininas que são reguladores centrais da atividade cambial. Considerando que no desenvolvimento da VB, a hiperplasia e hipertrofia de tecidos são sintomas típicos da doença, o envolvimento deste gene no processo pode ser relevante. O banco de dados gerados pelo sequenciamento, o conhecimento gerado sobre os mecanismos de defesa nos estágios iniciais da interação Cupuaçu - M. perniciosa e o estudo de função de genes envolvidos nesses mecanismos podem ser recursos valiosos para análises mais profundas, fornecendo maiores esclarecimentos sobre os mecanismos envolvidos na resistência e suscetibilidade em cupuaçuzeiros, subsidiando o desenvolvimento de programas de melhoramento e de estratégias de controle de VB.Theobroma grandiflorum is a fruit tree native to the Amazon region and a relative of cacao (T. cacao). It has a huge economic potential due to its multiple uses in the food and cosmetic industries. Cupuassu farming is seriously affected by Moniliophthora perniciosa, a fungus that causes witches' broom disease (WBD), in cupuassu, as well as in cacao. Knowledge about this phytopathosystem is essential for proposing strategies to control and mitigate the damage caused by WBD to these cultures. To provide insights into cupuassu resistance to M. perniciosa, the transcriptomic profiles of a resistant (clone 174) and a susceptible genotype (clone 1074) were analyzed using RNA-seq technology. In this study, we present the analyses of the transcriptome of both genotypes challenged with M. perniciosa, at different times of exposure to the pathogen, in the early stage of infection. A total of 21,441 unigenes and 440 differentially expressed genes (DEGs) were identified among the different conditions. The intrinsic difference analysis between the genotypes showed 301 DEGs. Gene expression alteration was observed earlier in the susceptible genotype at 24 hours after inoculation (hai). In the resistant one, the alteration was more prominent at 48 hai. These data set allowed the identification of genes potentially involved in the mechanism of defense, among them, pattern-recognition receptors (PRRs), transcription factors, pathogenesis related proteins (PRs), proteins related to cell wall remodelling, genes related to reactive oxygen species (ROS) accumulation and terpene pathways. The phytohormone signature analysis revealed a significant hormonal influence in genotypes’ responses. The genotype differed mainly relatively to auxin, cytokinin, salicylic acid and brassinosteroids responses. This is the first large-scale transcriptome study of T. grandiflorum, which in addition to providing insights into the resistance process, generated a list of potentially important genes in this process. Three genes from this list were selected for functional evaluation by heterologous expression in Micro-Tom (MT) tomato: 1) the transcription factor TgERF9, 2) the thaumatin-like protein (TgTLP1) and 3) TgPR10.1. The transformed plants, expressing the gene of interest, were challenged with hemibiotrophic phytopathogenic fungi (Fusarium oxysporum f.sp. lycopersici race 3, Verticillium dahliae race 2) and a necrotrophic one (Sclerotinia sclerotiorum). MT_TgERF9 and MT_TgPR10.1 plants were challenged with M. perniciosa. Furthermore, a study of the localization of TgPR10.1 transcripts in the tissues of the apical bud of cupuassu was carried out, via in situ hybridization (ISH). All fungal species were able to colonize plant tissues, either transformed or non-transformed plants. However, despite the darkening into the stem of plants inoculated with Verticillium and Fusarium, wilt symptoms were not observed, even in a water deficit condition. Furthermore, plant growth and fruit production were not affected by infection. Plants transformed with TgERF9 and TgTLP1 tended to be smaller and less productive. The detached leaf bioassays indicate that the expression of TgTLP1 increased the susceptibility of MT to the fungus S. sclerotiorum. MT_TgPR10.1 plants showed moderate resistance to S. sclerotiorum. Expression of this gene did not affect the development of WBD in MT. Nonetheless, MT_TgPR10.1 presented a height increase, indicating a change in hormonal balance. Regarding the location of the TgPR10.1 transcripts in cupuassu, it was possible to identify expression of this gene in the trichomes, in the procambium, in the meristem, and in the cells of the epidermis of the leaf primordia. The presence of these transcripts, particularly in the procambium, indicates a role for this PR in plant development and growth, which are affected by cytokinins, which are central regulators of cambial activity. Considering that in the development of witches' broom, tissue hyperplasia and hypertrophy are typical symptoms of the disease, the involvement of this gene in the process may be relevant. More detailed studies, such as measurements of phytohormones concentration, RNAseq and effects on endophytic organisms are necessary to better understand the function of these genes in the process of resistance and susceptibility to diseases. The database generated by sequencing, the insights about the defense mechanisms in the early stages of cupuassu- M. perniciosa interaction, and the study of the function of genes involved in these mechanisms can be a valuable resource for deeper analyses, which can help in the development of the cupuassu culture. These data can be a valuable resource for deeper analyses, providing further clarification of the mechanisms involved in resistance and susceptibility in cupuassu, supporting the development of its breeding programs and of strategies to control WBD.Instituto de Ciências Biológicas (IB)Departamento de Biologia Celular (IB CEL)Programa de Pós-Graduação em Biologia Molecula

Cupuassu (<i>Theobroma grandiflorum</i> [Willd. ex Sprengel] Schumann) Fruit Development: Key Genes Involved in Primary Metabolism and Stress Response

Cupuassu (Theobroma grandiflorum [Willd. ex Sprengel] Schumann) seeds constitute the raw material for oil extraction and fabrication of cupulate (product similar to chocolate). However, fungal diseases such as witches’ broom caused by Moniliophthora perniciosa have interfered with the large-scale development of cupuassu plantations. Cupuassu genetic breeding programmes focus on a variety of biotechnological tools or approaches to select genes related to quality or resistance mechanisms. In this study, we used expression and interactomics analyses of preselected genes involved in fruit quality and/or resistance to better understand the molecular and physiological mechanisms associated with these plant processes. It was found that (i) resistant and susceptible cupuassu genotypes showed different pulp characteristics as well as gene expression patterns; (ii) monosaccharide and carbohydrate transport pathways were enhanced during fruit maturation; (iii) sugar accumulation participated in signal transduction associated with fruit development and stress response in maturing fruits; and (iv) maturing pulp and seeds showed increased phospholipid metabolism and translocation, as well as immune system activation. The TgSTP1, TgWRKY33, TgCZF1, and TgUBA1 genes in cupuassu and the orthologues of DIN10, CNI1, and TET8 identified by the interactomics approach may be good candidates for marker-assisted selection in breeding programmes focusing on both fruit quality and resistance/tolerance to biotic/abiotic stress

Comparative transcriptomics of cupuassu (<i>Theobroma grandiflorum</i>) offers insights into the early defense mechanism to <i>Moniliophthora perniciosa</i>, the causal agent of witches’ broom disease

Cupuassu (Theobroma grandiflorum) is a fruit tree native to the Amazon region, presenting high social and economic value. Besides, owing to its suitability for agroforestry cultivation, cupuassu is useful for the conservation of the Amazon Forest. Cupuassu plantations are severely affected by Moniliophthora perniciosa. Thus, to gain insights into resistance against M. perniciosa, transcriptomes of susceptible (SG) and resistant (RG) genotypes of cupuassu were analyzed at the early stage of infection using RNA sequencing. A total of 21,441 unigenes were identified, and differentially expressed genes were detected in intra- (440) and inter-genotype (301) analyses. Gene expression was altered at 24 h after inoculation (HAI) in SG. This alteration was prominent at 48 HAI in RG. These datasets allowed the identification of genes potentially involved in defense mechanisms. Phytohormone signature analysis revealed a significant effect of hormones on genotype responses. The present study is the first large-scale transcriptomic analysis of cupuassu.</p

Candidate reference genes, primer characteristics, and efficiency obtained by RT-qPCR.

<p>CV: coefficient of variation, F: forward; R: reverse.</p

Best reference genes obtained by the BestKeeper program.

<p>SD: standard deviation; <i>r</i>: coefficient of correlation; CV: coefficient of variation. Best reference genes are indicated in bold.</p

Expression stability of the candidate reference genes obtained with NormFinder program.

<p>Data in bold indicates the best two genes for the sample group analyzed.</p