Biochemical, structural and functional analysis of CsMES1, a C. sinensis salicylate methylesterase involved in the defense response against citrus canker

Author(s)

Caio Cesar de Lima Silva

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-02-27

Examining board members

Celso Eduardo Benedetti; Alessandra Alves de Souza; Ana Carolina Migliorini Figueira; Priscila Oliveira de Giuseppe; Ronaldo José Durigan Dalio

• Advisor: Celso Eduardo Benedetti

Abstract

Salicylic acid (SA) and its methyl ester, methyl salicylate (MeSA), are known as inducers of local and systemic defense in plants, respectively. SA is important for the local defense response against biotrophic microorganisms, and its conversion from MeSA is essential in the development of systemic acquired resistance (SAR). This conversion is mediated by MeSA esterases of the MES/SABP2 family. Previous large-scale gene expression analyses of sweet orange leaves (Citrus sinensis) in response to infection by Citrus Canker pathogens have identified a gene encoding a protein belonging to the MES/SABP2 family, denominated CsMES1. CsMES1 is induced in response to Xanthomonas citri subsp. citri (Xc), the causal agent of Citrus Canker, but preferentially induced in response to Xanthomonas fuscans subsp. aurantifolii pathotype `C¿ (Xa), which induces a hypersensitive response in sweet oranges. Thus, the main objectives of this work were to evaluate the role played by CsMES1 in the local defense response against Xa and Xc, as well as to biochemically characterize its enzyme activity. In this work, we show that the Citrus sinensis methyl esterase CsMES1 is not only structurally related to tobacco Salicylic Acid-Binding Protein 2 (NtSABP2), but it also catalyzes the conversion of MeSA into SA. Structural molecular modeling indicates that CsMES1 and NtSABP2 share the same catalytic triad and bind SA in a similar fashion. Nonetheless, we found that CsMES1 has a lower affinity for SA compared to NtSABP2. Consistent with these findings, an amino acid polymorphism found in the active site of CsMES1-related proteins in C. sinensis and other plant species was shown to drastically affect the methylesterase activity and SA affinity in CsMES proteins. In addition, we provide evidence that the binding affinity between CsMES1 and SA is dependent on the ortho-hydroxyl group in the aromatic ring of SA. We also show that SA and transcripts of CsMES1 and other genes involved in SA signaling and response are detected in sweet orange leaves preferably during the incompatible interaction with Xa, compared with the compatible interaction with Xc. We have also shown that SA and MeSA applications inhibit the development of Canker symptoms caused by Xc, whereas a competitive inhibitor of CsMES1 increases the severity of Citrus Canker. These results thus suggest that CsMES1 and SA play an important role in local defense against Citrus Canker bacteria (AU)

• FAPESP's process: 13/22399-2 - Biochemical and functional analysis of a citrus sinensis protein, CsEIE, involved in the defense against Xanthomonas citri
• Grantee: Caio Cesar de Lima Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)