BASILA · Identification and characterization of bacterial effectors that interfere with Transcriptional Gene Silencing in Arabidopsis

Identification and characterization of bacterial effectors that interfere with Transcriptional GeneSilencing in Arabidopsis

The innate immune response is initiated by the recognition of either evolutionarily conserved pathogen-derived signatures known as Microbe-Associated Molecular Patterns (MAMPs) or the activity of virulence proteins secreted by pathogens called effectors, through PRR receptors or intracellular disease resistance (R) proteins, respectively. Both MAMP-triggered immunity (MTI) and effector-triggered immunity (ETI) rely on a massive transcriptional reprogramming, which is dependent on the accumulation and perception of signalling hormones and results in the differential expression of thousands of immune-responsive genes. Both MTI and ETI were found to be regulated at the post-transcriptional level by RNA silencing, a conserved eukaryotic mechanism that can negatively regulate gene expression in a sequence-specific manner via small RNA guides. RNA silencing also occurs at the transcriptional level through a chromatin-based regulatory mechanism referred to as RNA-dependent DNA methylation (RdDM), which silences repeated sequences and transposable elements to prevent their overexpression and proliferation. Transcriptional gene silencing (TGS) is an ancestral gene regulatory mechanism that operates at the level of chromatin and that is mediated by various histone modifications as well as cytosine DNA methylation. In both plants and animals, DNA methylation transcriptionally silences transposable elements (TEs), repeats, as well as some protein-coding genes that carry TEs/repeats in their vicinity. Notably, the expression of genes that contain transposon sequences in their vicinity can also be modulated by this epigenetic mechanism during their development or during biotic and abiotic stress responses. Numerous factors have been identified as critical components of RdDM, and many remain to be discovered- but this key pathway consists essentially in the production by DICER-Like 3 (DCL3) of 23-24 nt small interfering RNAs that are loaded onto ARGONAUTE 4 (AGO4) to guide DNA methylation. This siRNA-directed DNA methylation is catalyzed by Domain Rearranged Methyltransferase 2 (DRM2) at cytosines in all sequence contexts (CG, CHG and CHH where H is any nucleotide but not a G). During DNA replication, symmetric CG and CHG methylation are then maintained by Methyltransferase 1 (MET1) and the plant specific Chromomethylase 3 (CMT3), while asymmetric CHH methylation is actively perpetuated by RdDM or by CMT2 is a small RNA-independent manner. On the other hand, Arabidopsis encodes DNA glycosylases/lyases that can actively erase DNA methylation, among which Repressor Of Silencing 1 (ROS1), which has been extensively characterized as a negative regulator of RdDM. Whereas DNA methylation has been extensively studied mechanistically and during plant and developmental processes, its dynamics and biological relevance in response to biotic stresses, and particularly during bacterial infections, has just started to be examined and reported. Previous work from the host laboratory has revealed that the disease resistance gene RMG1 and potentially a large number of other targets involved in defence are directly controlled by DNA methylation and active DNA demethylation. This suggested that some bacterial virulence factors may have evolved to interfere with TGS to enable disease. The proposed project aimed at identifying and characterizing such bacterial effectors and their cellular targets. The project was primarily composed of three main tasks: WP1: Identification of the whole set of Pto DC3000 effectors that interfere with TGS WP2: Characterization of functionally relevant effector effects on TGS and innate immunity WP3: Identification and functional characterization of bacterial effector DNA/protein targets in host cells

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