[PhD Thesis Presentation] - Nino Espinas - rCBP-dependent regulation in rice innate immunity
Plant innate immunity against bacterial attacks is a two-tiered inducible system capable of defense responses at local and systemic areas. These systems are the PTI and ETI. During infection, PTI has the ability to recognize microbial signatures upon bacterial contact, while ETI recognizes microbial protein secretions called effectors delivered inside the cell. The activation of PTI and ETI confers systemic tissues of infected plants a broad-spectrum immunity against later pathogen attacks termed systemic acquired resistance (SAR). Defense priming is an adaptive component of SAR that regulates the molecular storage of defense memory for a more effective defense response.
The main aim of this work is finding a novel molecular defense signaling pathway that is controlled by acetylation at the local (basal defense) and systemic tissues (priming defense) and eventually during transgenerational transmission regulation.
To investigate the role of histone acetyltransferase-dependent pathway in basal defense, I have isolated transgenic and mutant lines of rCBP, [rice Cyclic adenosine monophosphate response element-binding protein (CREB) Binding Protein], under Nipponbare cultivar background using RNAi- and gRNA/Cas9-mediated genome editing. CBP was initially described as both transcriptional coactivator and histone acetyltransferase. The rCBP-RNAi lines with mistargeting of the other members of CBP family are characterized by massive sterility and impairment of the number of effective grains. On the other hand, the CRISPR/Cas9 mutant lines have wild-type number of effective grains.
To profile the global acetylation of histone lysine-sites via rCBP, I performed mass spectrometry-based proteomics in data dependent acquisition (DDA) and parallel reaction monitoring (PRM) modes. My results showed that H3 lysine sites are mostly targeted by rCBP with very high acetylation specificity on H3K9.
To implicate the role of rCBP in rice innate immunity, I conducted an enhanced disease susceptibility (EDS) assay. EDS assay showed that rCBP-/- mutants are resistant to pathogen infection compared to segregated wild-type control.
I also performed RNA-sequencing on locally-infected and systemic tissues to investigate the genome-wide effects of rCBP mutation and to identify factors with roles in both basal and systemic immune response. As a result, I have identified seven putative rCBP-dependent repressor transcription factors that possibly explain the resistance phenotype of mutant lines.
Overall, these data preliminary indicate that rCBP is both a positive regulator of developmental processes and a negative regulator of rice immunity. Our data also suggest that rCBP possibly executes this dual regulatory function mostly through H3K9ac and associated regulatory protein networks on target gene loci.