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Functional characterization of a chitinase-like protein of Moniliophthora perniciosa with a potential role in fungal pathogenicity during the development of the witches' broom disease of cacao

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Gabriel Lorencini Fiorin
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
Gonçalo Amarante Guimarães Pereira; Fabio Papes; Antonio Vargas de Oliveira Figueira
Advisor: Gonçalo Amarante Guimarães Pereira; Paulo José Pereira Lima Teixeira

Cacao (Theobroma cacao) is a perrenial crop of remarkable economic importance worldwide, historically accounting for wealth to Brazil. The productivity of brazilian cacao farming suffered a drastic decrease over the 90¿s, when the witches¿s broom disease (WBD), caused by the basidiomycete Moniliophthora perniciosa, spread widely throughout the major brazilian producing region, southern Bahia. The impact of this severe disease is bringing economic, social and environmental issues to the country, which makes the development of efficient management strategies a clear priority. Important progress has been achieved in understanding the pathogenicity mechanisms deployed by M. perniciosa during plant colonization through genomic and transcriptomic approaches. Remarkably, a first inspection of gene expression data revealed a fungal chitinase (Mp-chi) among the genes highly and exclusively expressed during the biotrophic stage of cacao infection. Surprisingly, though, the predicted amino acid sequence of such chitinase exhibited two substitutions (E167?Q and M238?L) in key conserved residues for enzymatic catalysis, which presumably would lead to complete absence of chitinolytic activity. Taken together, these properties suggested that the chitinase Mp-Chi, instead of participating in chitin catabolism, could be playing a role in fungal pathogenicity. Given that the residues involved in chitin binding are conserved in the protein sequence, its role in the interaction could be related to chitin binding. In order to investigate the functional role performed by Mp-Chi during WBD, the protein was expressed in heterologous system and subjected to in vitro functional assays. The results showed that Mp-Chi is indeed catalytically inactive. The enzymatic function of Mp-Chi was regained upon restoration of both conserved catalytic residues, demonstrating that the absence of chitinolytic activity is a direct consequence of these two substitutions, at least. Carbohydrate binding assays demonstrated that Mp-Chi binds chitin and chito-oligomers (GlcNAc6), which would enable the protein to act as a fungal pathogenicity tool, either protecting the fungal cell wall from hydrolytic enzymes secreted by the host plant or suppressing Chitin-Triggered Immunity. Using model experimental platforms, it was demonstrated that, although Mp-Chi does not provide protection against hydrolytic enzymes, the protein prevents the activation of Chitin-Triggered Immunity. Remarkably, the ability to circumvent Chitin- Triggered Immunity was abolished in a mutated version of the protein impaired in chitin binding, indicating that the ability to bind chitin is the key functional property for Mp-Chi to play its role in fungal pathogenicity. Collectively, the results support the role of Mp-Chi as a potential effector protein deployed by M. perniciosa to avoid recognition by the host plant, therefore contributing for successful colonization of T. cacao. The functional characterization of Mp-Chi represents an important step towards a better understanding of the evasion strategies against the host plant exploited by M. perniciosa during WBD. Also, given that a chitinase has never been implicated in such function, our findings represent a relevant novelty in modern plant pathology. In addition to the functional characterization of Mp-Chi, we started the development of a methodology for M. pernciosa genetic manipulation using the novel CRISPR/Cas9 technology. The vector containing all the elements required for genome editing has been built and will allow fungal transformation. Once established, the procedure will allow for the production of Mp-chi deficient strains and several other strains of interest, providing a powerful tool for understanding the mechanisms involved in M. perniciosa pathogenicity in vivo (AU)

FAPESP's process: 14/06181-0 - Development of a CRISPR/Cas9-based methodology for genome engineering in the fungus Moniliophthora perniciosa, the causative agent of witches' broom disease of cacao
Grantee:Gabriel Lorencini Fiorin
Support type: Scholarships in Brazil - Master