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SummaryBacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo).We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence.The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence.Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.

African strains of Xanthomonas oryzae pv. oryzae contain fewer TAL effectors than Asian strains, and their contribution to pathogenicity is unknown. Systematic mutagenesis of tal genes was used to decipher the contribution of each of the eight TAL effector paralogs to pathogenicity of African X. oryzae pv. oryzae BAI3. A strain mutated in talC was severely affected in the production of disease symptoms. Analysis of growth in planta upon leaf-clip inoculation showed that mutant bacteria multiplied only at the site of inoculation at the apex of the leaf, suggesting a requirement for talC during colonization of vascular tissues. Such tissue-specific effect of a tal mutant is a novel phenotype, which has not yet been characterized in other xanthomonads. Microarray experiments comparing the host response of rice leaves challenged with BAI3(R) vs. BAI3(R)ΔtalC were performed to identify genes targeted by TalC. A total of 120 upregulated and 21 downregulated genes were identified, among them Os11N3, which is a member of the MtN3/saliva family. Based on semiquantitative reverse transcription-polymerase chain reaction and β-glucuronidase reporter assays, we show that Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence.

SUMMARYGenome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T 0 and Arabidopsis T 2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.