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Publications - Cell and Metabolic Biology
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Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.
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The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. The T3S apparatus spans both bacterial membranes and is associated with an extracellular pilus and a channel-like translocon in the host plasma membrane. T3S is controlled by the switch protein HpaC, which suppresses secretion and translocation of the predicted inner rod protein HrpB2 and promotes secretion of translocon and effector proteins. We previously reported that HrpB2 interacts with HpaC and the cytoplasmic domain of the inner membrane protein HrcU (C. Lorenz, S. Schulz, T. Wolsch, O. Rossier, U. Bonas, and D. Büttner, PLoS Pathog 4:e1000094, 2008, http://dx.doi.org/10.1371/journal.ppat.1000094). However, the molecular mechanisms underlying the control of HrpB2 secretion are not yet understood. Here, we located a T3S and translocation signal in the N-terminal 40 amino acids of HrpB2. The results of complementation experiments with HrpB2 deletion derivatives revealed that the T3S signal of HrpB2 is essential for protein function. Furthermore, interaction studies showed that the N-terminal region of HrpB2 interacts with the cytoplasmic domain of HrcU, suggesting that the T3S signal of HrpB2 contributes to substrate docking. Translocation of HrpB2 is suppressed not only by HpaC but also by the T3S chaperone HpaB and its secreted regulator, HpaA. Deletion of hpaA, hpaB, and hpaC leads to a loss of pathogenicity but allows the translocation of fusion proteins between the HrpB2 T3S signal and effector proteins into leaves of host and non-host plants.IMPORTANCE The T3S system of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is essential for pathogenicity and delivers effector proteins into plant cells. T3S depends on HrpB2, which is a component of the predicted periplasmic inner rod structure of the secretion apparatus. HrpB2 is secreted during the early stages of the secretion process and interacts with the cytoplasmic domain of the inner membrane protein HrcU. Here, we localized the secretion and translocation signal of HrpB2 in the N-terminal 40 amino acids and show that this region is sufficient for the interaction with the cytoplasmic domain of HrcU. Our results suggest that the T3S signal of HrpB2 is required for the docking of HrpB2 to the secretion apparatus. Furthermore, we provide experimental evidence that the N-terminal region of HrpB2 is sufficient to target effector proteins for translocation in a nonpathogenic X. campestris pv. vesicatoria strain.
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