Functional analysis of the cyclophilin CsCyp on citrus genes transcription activated by tal effectors from Xanthomonas citri

• Grant number: 14/19623-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: December 01, 2014
• End date: November 30, 2016
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Celso Eduardo Benedetti
• Grantee: Paula Rodrigues Oblessuc
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 11/20468-1 - Molecular mechanisms involved in pathogen adaptation and virulence, host resistance and symptom development in citrus-bacteria interactions, AP.TEM

Abstract

The citrus canker disease, caused by the bacteria Xanthomonas citri, affects most commercial citrus varieties, and in recent years, its incidence has increased significantly in the main citrus producing regions of the country. Aiming to understand how X. citri induces cell growth and expansion in citrus, the typical canker symptoms, our group identified several proteins from Citrus sinensis that interact with the effector protein PthA, the main virulence factor of X. citri. PthAs belong to the TAL (Transcription activator-like) effectors family, which bind specific promoter sequences and activate transcription of target genes in the plant. Among the citrus proteins that interact with PthAs, we identified the cyclophilin (prolyl isomerase) CsCyp, CsSUMO (small ubiquitin-related modifier), and the RNA polymerase II C-terminal domain (CTD). Furthermore, gene expression analysis and in silico identification of citrus gene promoter regions that contain PthAs binding sites allowed us to identify potential PthAs target genes; including the Lateral Organ Boundary 1 (CsLOB1) gene. We also found that PthA2 inhibit the prolyl isomerase activity of CsCyp and citrus plants with reduced levels of CsCyp were more susceptible to X. citri. In addition, CsCyp interacted with the citrus CTD and also complemented the yeast mutants deficient in the prolyl isomerases Cpr1 and Ess1, which isomerize proline residues of the CTD and control elongation and termination of transcription in yeast. Therefore, we hypothesize that PthAs activate transcription of target genes by modulating the activity of the CTD via interaction with CsCyp. The objectives of this proposal include: (1) confirm the direct interaction of PthAs with the promoter region of target genes, especially CsLOB1; (2) evaluate the expression of PthAs target genes in citrus transgenic lines with altered levels of CsCyp by overexpression and RNAi; (3) verify the interaction of CsCyp with the chromatin in the promoter, coding and/or terminator regions of PthA target genes; (4) evaluate whether phosphorylation of serines 2 and 5 of the citrus CTD repeats affects the interaction with CsCyp; and (5) verify if CsCyp is sumoylated during the development of canker lesions. We believe this work will enable a better understanding of the role of CsCyp in the transcriptional control mediated by PthAs. (AU)