Identification and characterization of host targets of Ralstonia solanacearum TAL effector Brg11

Ralstonia solanacearum is a devastating bacterial plant pathogen that causes bacterial wilt disease across a wide range of plant species including numerous important crops. During infection, R. solanacearum injects effector proteins into host cells that interfere in various ways with cellular processes to promote disease. Brg11 is an effector present in the R. solanacearum strain GMI1000 that shares homology to transcription activator-like effectors (TALEs) from Xanthomonas. TALEs bind to effector binding elements (EBEs) in host promoters and transcriptionally activate downstream host genes to either promote disease or trigger resistance. Previous in planta studies revealed that the TALE-like Brg11 protein transcriptionally activates reporter genes that contain a compatible EBE in their promoters. However, the plant target(s) of Brg11 and their contribution to bacterial virulence is still unclear. I started my work by establishing a modular, Golden-Gate (GG)-based toolkit that simplifies assembly of R. solanacearum gene knockout and complementation constructs. This toolkit contains a series of vector backbones and functional modules that are all compatible with a previously established GG-toolkit for in planta gene expression. This toolkit will foster genetic studies in R. solanacearum. In the second part of my work I identified Brg11 host target genes by the combined use of transcriptome profiling and target site prediction. This work uncovered, that R. solanacearum Brg11 transcriptionally activates exclusively tomato (Solanum lycopersicum) arginine decarboxylase 1 and 2 genes (SlADC1/2). Mutational studies demonstrate that Brg11-dependent activation of SlADC1/2 relies on integrity of Brg11 target sites (Brg11-EBEs) that are located upstream of the SlADC1/2 coding sequences. Inspection of R. solanacearum plant host genomes shows in each case at least one ADC gene with a Brg11 compatible EBE upstream of the ADC coding sequence. Quantification of ADC transcript levels demonstrated Brg11-dependent activation in eggplant, Nicotiana benthamiana and N. tabacum. In summary these observations suggest that ADC genes are conserved targets of Brg11 across phylogenetically distinct plant hosts of R. solanacearum. The third part of my work focused on the molecular characterization of Brg11-induced ADC transcripts. 5’RACE PCR revealed that Brg11 induces ADC transcripts with 5’UTRs that are ~300 bp shorter than native ADC transcripts. Comparison of translational activity of native vs. Brg11-induced, short ADC transcripts revealed that the latter have higher translational activity. Mutational studies revealed that a conserved ~50 bp GC-rich motif in the 5’UTR of native ADC transcripts, termed ADC- box, attenuates translation possibly by forming a RNA secondary structure. Since Brg11-induced ADC transcripts have a short 5’UTR lacking the ADC-box these transcripts bypass translational regulation installed by the host, resulting in high ADC expression. How Brg11-mediated ADC activation contributes to R. solanacearum virulence was studied in part four of this work. ADC proteins are known to catalyze conversion of arginine to agmatine and are rate-limiting enzymes of polyamine biosynthesis. Metabolic studies revealed a Brg11-dependent increase of agmatine and diamine putrescine but not of the polyamines spermidine and spermine. Elevated putrescine is often associated with plant defense reactions. Quantification of in planta growth indeed revealed that the Brg11-induced increase in putrescine levels correlates with reduced in planta growth of Pseudomonas syringae pv. tomato DC3000 but notably had no impact on growth of R. solanacearum. These observations suggest a model where Brg11-mediated increase of host putrescine results in reduced growth of microbial niche competitors of R. solanacearum. Observations of this work suggest a novel concept in which type III effectors mediate ternary microbe-host-microbe interactions to promote bacterial virulence. This work also suggests polyamine biosynthesis as a yet unrecognized regulatory hub that is manipulated by microbial type III effectors. Furthermore this work resulted in the identification of the ADC-box, a cis element present in 5’UTRs of land plant ADC transcripts that controls ADC expression levels and thus serves as a regulator of polyamine biosynthesis. These findings provide opportunities for molecular breeding of pest-resistant plants