Analysis of TALE repeats of aberrant length and identification of novel candidate target genes for Xanthomonas oryzae pv. oryzicola TALEs

Abstract

Transcription activator-like effectors (TALEs) are bacterial proteins most prominently found in plant pathogenic Xanthomonas species. TALEs mimic the function of eukaryotic transcription factors in order to upregulate the expression of their plant target genes. Their unique structure, an array of consecutively placed 33-35 amino acids long repeats, allows them to bind to DNA in a simple one repeat to one nucleotide manner. However, a few naturally occurring repeat-variants contain small duplications or deletions whose presence enables a TALE to break with this strict one repeat to one nucleotide binding mechanism, allowing for the recognition of an additional target sequence with a -1 nucleotide frameshift. In this work, these aberrant repeats were analysed in detail, demonstrating that a single TALE can tolerate more than one or two aberrant repeats by simultaneously looping out multiple repeats. It was also shown that two aberrant repeats need a minimal distance to act functionally independent and that they allow a TALE, if properly spaced, to flexibly recognize a multitude of different frameshift sequences. Novel natural aberrant repeat variants were identified and analysed, revealing that not all of them confer frameshift binding. Two variants did not alter the DNA-binding of the tested TALE while one variant was unable to participate in DNA-binding. By investigating a set of artificial aberrant repeats, it was found that the nature as well as the position of the altered amino acids influence the functionality of aberrant repeats. This analysis also allowed to visualize the tolerance of a standard TALE repeat to small insertions and resulted in an experiment that could show that it is possible to insert complete functional domains from other proteins between two TALE repeats. Furthermore, the first natural TALE to contain two aberrant repeats was identified and its ability to bind to multiple frameshift sequences was demonstrated. It was also shown that this TALE targets an allele of the susceptibility gene OsSWEET13 from the rice cv. Nipponbare. In addition, a newly identified OsSWEET13 allele from the cv. Sadu Cho was shown to confer resistance against both known TALEs addressing OsSWEET13. Recent technological advancements lead to an increased availability of X. oryzae genomes and TALE sequences alike. Despite numerous efforts, for many of these TALEs, no plant target gene is known. To address this issue, most of the naturally occurring X. oryzae pv. oryzicola TALEs were artificially re-constructed. In subsequent GUS reporter studies seven genes previously predicted as TALE targets based on RNA-Seq data were confirmed experimentally. De novo TALE target predictions led also to the identification of 22 novel TALE target gene candidates whose promoters were directly induced by a Xoc TALE.