Título:	Pepper ringspot virus : estudo da resposta imune em Nicotiana benthamiana e utilização como vetor para produção de antígeno específicos contra Coguvírus
Outros títulos:	Pepper ringspot virus : study of the immune response in Nicotiana benthamiana and use as a vector for production of specific antigen against Coguvirus virus
Autor(es):	Kauffmann, Caterynne Melo
Orientador(es):	Nagata, Tatsuya
Assunto:	Coguvirus Melancia Nicotiana benthamiana Imunidade inata Morte celular Clone infeccioso
Data de publicação:	16-Dez-2025
Data de defesa:	21-Fev-2025
Referência:	KAUFFMANN, Caterynne Melo. Pepper ringspot virus: estudo da resposta imune em Nicotiana benthamiana e utilização como vetor para produção de antígeno específicos contra Coguvírus. 2025. 98 f., il. Tese (Doutorado em Fitopatologia) — Universidade de Brasília, Brasília, 2025.
Abstract:	The viruses Coguvirus citrulli and C. henanense, respectively known as watermelon crinkle leaf-associated virus (WCLaV-1 and WCLaV-2), respectively, belong to the genus Coguvirus and were recently detected in watermelon plants in Brazil, frequently co-infecting plants alongside Orthotospovirus arachianuli (groundnut ringspot virus, GRSV). Plants infected with WCLaVs and GRSV exhibit symptoms such as mosaic, leaf curling, and lamina reduction. Despite the severity of these symptoms, limited information is available on the epidemiology of the disease in Brazil, including yield losses, alternative hosts, and transmission vectors. To address these gaps, the development of sensitive and specific detection tools for these viruses is essential. This study aimed to develop specific polyclonal antibodies against WCLaV-1 and WCLaV-2. The approach involved the production of nucleocapsid (N) proteins of WCLaV-1 and WCLaV-2 using pepper ringspot virus (PepRSV) as a viral vector in Nicotiana benthamiana for heterologous expression. The recombinant proteins were injected into rabbits, and the resulting antisera were evaluated for specificity using dot-immunobinding assay (DIBA), antigen-coating ELISA, tissue blot immunobinding assay (TBIA), and Western blot. Additionally, ELISA analyses indicated that WCLaV-1 exhibited a 93.1% seed infection rate in commercial seeds of the 'Crimson Sweet' cultivar, whereas WCLaV2 was detected in only 17.8% of samples. TBIA analysis confirmed the presence of WCLaV-1 in seeds and seedlings, and confocal microscopy revealed that the N protein of WCLaV-1 was predominantly localized in the cytoplasm of seed tissues, with additional detection in the nuclei of root-tip cells. Infectious clones of PepRSV, composed of two genomic segments (RNA1 and RNA2), were cloned into the binary vector pJL89. Agroinfiltration of these infectious clones via Agrobacterium tumefaciens in N. benthamiana resulted in systemic infection, enabling the use of PepRSV as a viral vector for heterologous protein expression. This system allows for the replacement of the PepRSV coat protein (CP) gene with a gene of interest, facilitating, for example, the heterologous expression of a foreign CP in infected plants. During experiments with the PepRSV infectious clone, a mutant of RNA2, designated clone 4, was identified. This mutant carried an atypical mutation characterized by the insertion of a cytidine at the 3' end of the CP gene. Inoculation of clone 4 into N. benthamiana resulted in the formation of local necrotic lesions resembling a hypersensitive response (HR-like). In contrast, another RNA2 clone, designated clone 2, retained the wild-type genomic sequence and induced systemic infection without local lesions. Given the significance of this finding, further studies were conducted to elucidate the mechanism underlying the HR-like response induced by clone 4. The experiments aimed to evaluate the immune response of N. benthamiana infiltrated with clone 4, focusing on the activation of reactive oxygen species, particularly hydrogen peroxide accumulation, callose deposition, expression of defense-related genes via RT-qPCR, and the impact of SGT1 gene silencing. The results showed that plants inoculated with the clone 4 exhibited both hydrogen peroxide accumulation and callose deposition, whereas plants infected with the clone 2 displayed only callose deposition. Additionally, the expression of defense marker genes was significantly higher in plants infected with the clone 4, and SGT1 gene silencing was detected. However, further analyses are required to clarify the mechanisms involved and determine whether the defense response is primarily mediated by pattern-triggered immunity (PTI) or effector-triggered immunity (ETI). The findings of this study contribute to the diagnosis and epidemiological understanding of WCLaVs in watermelon cultivation, highlighting seed transmission as a relevant factor. Furthermore, they advance the application of PepRSV as a viral vector for antigen expression and the investigation of immune responses in N. benthamiana. The characterization of PepRSV clone 4 enhances our understanding of plant defense mechanisms, enabling new approaches for studying virus-plant interactions and developing control strategies.
Unidade Acadêmica:	Instituto de Ciências Biológicas (IB) Departamento de Fitopatologia (IB FIT)
Informações adicionais:	Tese (doutorado) — Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, 2025.
Programa de pós-graduação:	Programa de Pós-Graduação em Fitopatologia
Licença:	A concessão da licença deste item refere-se ao termo de autorização impresso assinado pelo autor com as seguintes condições: Na qualidade de titular dos direitos de autor da publicação, autorizo a Universidade de Brasília e o IBICT a disponibilizar por meio dos sites www.bce.unb.br, www.ibict.br, http://hercules.vtls.com/cgi-bin/ndltd/chameleon?lng=pt&skin=ndltd sem ressarcimento dos direitos autorais, de acordo com a Lei nº 9610/98, o texto integral da obra disponibilizada, conforme permissões assinaladas, para fins de leitura, impressão e/ou download, a título de divulgação da produção científica brasileira, a partir desta data.
Agência financiadora:	Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Aparece nas coleções:	Teses, dissertações e produtos pós-doutorado

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